Novel EGFR-Binding Molecules and Immunoconjugates Thereof

ABSTRACT

Novel anti-cancer agents, including, but not limited to, antibodies and immunoconjugates, that bind to EGFR are provided. Methods of using the agents, antibodies, or immunoconjugates, such as methods of inhibiting tumor growth are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/408,497, filed Oct. 29, 2010, and U.S. ProvisionalApplication No. 61/477,086, filed Apr. 19, 2011, each of which is herebyincorporated by reference herein in its entirety

FIELD OF THE INVENTION

The present invention generally relates to antibodies, antigen-bindingfragments thereof, polypeptides, and immunoconjugates that bind to EGFR.The present invention also relates to methods of using such EGFR-bindingmolecules for diagnosing and treating diseases, such as malignancies.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor (EGFR or ErbB1 or HER1) is atransmembrane glycoprotein of 170 kDa that is encoded by the c-erbB1proto-oncogene located in the 7q22 chromosome. EGFR is a member of thehuman epidermal growth factor receptor (HER) family of receptor tyrosinekinases (RTK) which includes HER2 (ErbB2), HER3 (ErbB3) and HER4(ErbB4). These RTKs share a homologous structure that consists of aligand-binding extracellular domain (ECD), a single span transmembranedomain and an intracellular domain that contain catalytic-kinase domainand a C-terminal tail. HER kinase signaling pathways are initiated bythe binding of extracellular ligand that induces receptorhomodimerization or heterodimerization with other HER kinase member andtransphosphorylation of the intracellular regions. These events generatethe initial signal leading to activation of numerous downstreamsignaling pathways that are critical for cell proliferation andsurvival.

EGFR is over-expressed in many malignant tumor types of epithelial cellorigin such as head and neck, colorectal, lung, ovarian, renal,pancreatic, skin and other solid tumors. EGFR-mediated signalingpathways play a significant role in the progression of tumor growth andmetastases, making EGFR a good target for tumor therapy (Baselga,Oncologist, 7:2-8 (2002), Yarden and Sliwkowski, Nat Rev Mol Cell Biol,2:127-137 (2001)). At present, four EGFR targeting agents including twosmall molecules tyrosine kinase inhibitors (TKIs) (erlotinib (Tarcevafrom Genentech and OSI Pharmaceuticals) and gefitinib (Iressa fromAstraZeneca and Teva Pharmaceuticals)) and two naked monoclonalantibodies cetuximab (Erbitux from ImClone and BMS) and panitumumab(Vectibix from Amgen)) have been approved for treatment of colorectalcancer, pancreatic cancer, head and neck cancer, and non-small cell lungcancer (NSCLC). These anti-EGFR agents strongly inhibit EGFR activationand downstream signaling. The TKIs compete with ATP for binding to theEGFR's intracellular kinase domain (Baselga and Arteaga, J Clin Oncol,23:2445-2459 (20005)), whereas the two monoclonal antibodies competewith the EGFR ligands for binding to the receptor (Gill et al., J BiolChem, 259:7755-7760 (1984), Goldstein et al., Clin Cancer Res,1:1311-1318 (1995), Prewett et al., Clin Cancer Res, 4:2957-2966(1998)).

Anti-EGFR therapies are not perfect. Inhibition of EGFR signaling isonly effective in certain tumor type. For example, the efficacy ofanti-EGFR antibodies is significantly reduced in colorectal cancerpatients with KRAS, BRAF, PIK3CA and PTEN mutations (De Roock et al.,Lancet Oncol, 11:753-762 (2010), Bardelli and Sienna, J Clin Oncol, 28:1254-1261 (2010)). Additionally, the activity of small molecule EGFRinhibitors is limited to NSCLC patients with activating EGFR mutations(Linardou et al., Nat Rev Clin Oncol, 6: 352-366 (2009), Paz-Ares etal., J Cell Mol Med, 14: 51-69 (2009), Mok et al., Discov Med, 8:227-231 (2009)). EGFR therapies also result in skin toxicity. EGFRexpression in normal basal epithelial cells of the skin plays a crucialrole in normal development and physiology of epidermis, and inhibitionof EGFR signaling causes various skin toxicities including acneiformskin rash, skin dryness, pruritus, paronychia, hair abnormality,mucositis and increased growth of the eyelashes or facial hair (reviewedin L1 and Perez-Soler, Targ Oncol 4:107-119 (2009)). Although rarelylife-threatening, the skin toxicities cause significant physical andpsycho-social discomfort that decrease the patient's life quality.Additionally, in around 10% of patients, the skin toxicity is so severethat it requires treatment interruption or discontinuation that impairsthe clinical outcomes of EGFR inhibitors.

Accordingly, the need exists for improved anti-EGFR therapy which isharmless to normal tissues but still very effective in treating EGFRoverexpressing malignant tumors, in particular, tumors that areresistant to the current EGFR therapies. To address this particularneed, the present invention focuses on a unique novel class of EGFRantibodies that are very potent in killing EGFR expressing tumor cellsbut have no or little impact on normal epithelial cell growth.Furthermore, while the conjugation of the EGFR antibodies of theinvention with cytotoxic agents potentiates the anti-tumor activity ofthese antibodies, it does not cause additional toxicity to normalepithelial cells.

BRIEF SUMMARY OF THE INVENTION

The present invention generally relates to antibodies, antigen-bindingfragments thereof, polypeptides, and immunoconjugates that bind to EGFR.The present invention also relates to methods of using such EGFR-bindingmolecules for diagnosing and treating diseases, such as malignancies.

Thus, in one embodiment the invention provides an antibody or antigenbinding fragment thereof that specifically binds to human EGFR, whereinsaid antibody has at least one characteristic selected from the groupconsisting of: (a) inhibits at least 80% of epidermal growth factor(EGF) and transforming growth factor alpha (TGFα) binding to A431 cellsat a concentration of 10 nM or higher, (b) causes at least 50%inhibition of H292 and HCC827 tumor cell proliferation at 30 nM orhigher, and (c) does not inhibit more than 20% proliferation ofkeratinocytes and MCF-10A epithelial cells at 60 nM or lower. In anotherembodiment, the antibody has at least two characteristics selected fromthe group consisting of: (a) inhibits at least 80% of EGF and TGFαbinding to A431 cells at a concentration of 10 nM or higher, (b) causesat least 50% inhibition of H292 and HCC827 tumor cell proliferation at30 nM or higher, and (c) does not inhibit more than 20% proliferation ofkeratinocytes and MCF-10A epithelial cells at 60 nM or lower. In anotherembodiment, the antibody or antigen binding fragment thereof thatspecifically binds to human EGFR, wherein said antibody (a) inhibits atleast 80% of EGF and TGFα binding to A431 cells at a concentration of 10nM or higher, (b) causes at least 50% inhibition of H292 and HCC827tumor cell proliferation at 30 nM or higher, and (c) does not inhibitmore than 20% proliferation of keratinocytes and MCF-10A epithelialcells at 60 nM or lower.

In one embodiment, the invention provides an antibody which comprises(a) a VH sequence at least 90% identical to a reference VH sequenceselected from the group consisting of SEQ ID NOs:19-23 and 69-73; and(b) a VL sequence at least 90% identical to a reference VL sequenceselected from the group consisting of SEQ ID NOs:24-30 and 70. Inanother embodiment, the VH and VL sequences are at least 95% identicalto the reference VH and VL sequences. In another embodiment, the VH andVL sequences are at least 99% identical to the reference VH and VLsequences.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof which comprises (a) a VH sequence selected from thegroup consisting of SEQ ID NOs:19-23 and 69-73; and (b) a VL sequenceselected from the group consisting of SEQ ID NOs:24-30 and 70. Inanother embodiment, the antibody or antigen binding fragment thereofcomprises SEQ ID NO:19 and SEQ ID NO:24. In another embodiment, theantibody or antigen binding fragment thereof comprises SEQ ID NO:20 andSEQ ID NO:25. In another embodiment, the antibody or antigen bindingfragment thereof comprises SEQ ID NO:21 and SEQ ID NO:26. In anotherembodiment, the antibody or antigen binding fragment thereof comprisesSEQ ID NO:21 and SEQ ID NO:27. In another embodiment, the antibody orantigen binding fragment thereof comprises SEQ ID NO:22 and SEQ IDNO:28. In another embodiment, the antibody or antigen binding fragmentthereof comprises SEQ ID NO:23 and SEQ ID NO:29. In another embodiment,the antibody or antigen binding fragment thereof comprises SEQ ID NO:23and SEQ ID NO:30. In another embodiment, the antibody or antigen bindingfragment thereof comprises SEQ ID NO:69 and SEQ ID NO:70. In anotherembodiment, the antibody or antigen binding fragment thereof comprisesSEQ ID NO:71 and SEQ ID NO:26. In another embodiment, the antibody orantigen binding fragment thereof comprises SEQ ID NO:71 and SEQ IDNO:27. In another embodiment, the antibody or antigen binding fragmentthereof comprises SEQ ID NO:72 and SEQ ID NO:26. In another embodiment,the antibody or antigen binding fragment thereof comprises SEQ ID NO:72and SEQ ID NO:27. In another embodiment, the antibody or antigen bindingfragment thereof comprises SEQ ID NO:73 and SEQ ID NO:26. In anotherembodiment, the antibody or antigen binding fragment thereof comprisesSEQ ID NO:73 and SEQ ID NO:27.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof produced by hybridoma selected from the groupconsisting of ATCC Deposit Designation PTA-11331, deposited with theATCC on Oct. 6, 2010, ATCC Deposit Designation PTA-11332, deposited withthe ATCC on Oct. 6, 2010, and ATCC Deposit Designation PTA-11333,deposited with the ATCC on Oct. 6, 2010. In another embodiment, theinvention provides an antibody or antigen binding fragment thereof thatspecifically binds to the same EGFR epitope as an antibody selected fromthe group consisting of ATCC Deposit Designation PTA-11331, depositedwith the ATCC on Oct. 6, 2010, ATCC Deposit Designation PTA-11332,deposited with the ATCC on Oct. 6, 2010, and ATCC Deposit DesignationPTA-11333, deposited with the ATCC on Oct. 6, 2010. In yet anotherembodiment, the invention provides an antibody or antigen bindingfragment thereof that competitively inhibits binding of a referenceantibody to human EGFR, wherein said reference antibody is selected fromthe group consisting of ATCC Deposit Designation PTA-11331, depositedwith the ATCC on Oct. 6, 2010, ATCC Deposit Designation PTA-11332,deposited with the ATCC on Oct. 6, 2010, and ATCC Deposit DesignationPTA-11333, deposited with the ATCC on Oct. 6, 2010.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that specifically binds to the same EGFR epitope as anantibody selected from the group consisting of: (a) an antibodycomprising the VH polypeptide of SEQ ID NO:19 and the VL polypeptide ofSEQ ID NO:24; (b) an antibody comprising the VH polypeptide of SEQ IDNO:20 and the VL polypeptide of SEQ ID NO:25; (c) an antibody comprisingthe VH polypeptide of SEQ ID NO:21 and the VL polypeptide of SEQ IDNO:26; (d) an antibody comprising the VH polypeptide of SEQ ID NO:21 andthe VL polypeptide of SEQ ID NO:27; (e) an antibody comprising the VHpolypeptide of SEQ ID NO:22 and the VL polypeptide of SEQ ID NO:28; (0an antibody comprising the VH polypeptide of SEQ ID NO:23 and the VLpolypeptide of SEQ ID NO:29; (g) an antibody comprising the VHpolypeptide of SEQ ID NO:23 and the VL polypeptide of SEQ ID NO:30; (h)an antibody comprising the VH polypeptide of SEQ ID NO:69 and the VLpolypeptide of SEQ ID NO:70; (i) an antibody comprising the VHpolypeptide of SEQ ID NO:71 and the VL polypeptide of SEQ ID NO: 26; (j)an antibody comprising the VH polypeptide of SEQ ID NO:71 and the VLpolypeptide of SEQ ID NO:27; (k) an antibody comprising the VHpolypeptide of SEQ ID NO:72 and the VL polypeptide of SEQ ID NO:26; (l)an antibody comprising the VH polypeptide of SEQ ID NO:72 and the VLpolypeptide of SEQ ID NO:27; (m) an antibody comprising the VHpolypeptide of SEQ ID NO:73 and the VL polypeptide of SEQ ID NO:26; and(n) an antibody comprising the VH polypeptide of SEQ ID NO:73 and the VLpolypeptide of SEQ ID NO:27.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that competitively inhibits binding of a referenceantibody to human EGFR, wherein said reference antibody is selected fromthe group consisting of: (a) an antibody comprising the VH polypeptideof SEQ ID NO:19 and the VL polypeptide of SEQ ID NO:24; (b) an antibodycomprising the VH polypeptide of SEQ ID NO:20 and the VL polypeptide ofSEQ ID NO:25; (c) an antibody comprising the VH polypeptide of SEQ IDNO:21 and the VL polypeptide of SEQ ID NO:26; (d) an antibody comprisingthe VH polypeptide of SEQ ID NO:21 and the VL polypeptide of SEQ IDNO:27; (e) an antibody comprising the VH polypeptide of SEQ ID NO:22 andthe VL polypeptide of SEQ ID NO:28; (f) an antibody comprising the VHpolypeptide of SEQ ID NO:23 and the VL polypeptide of SEQ ID NO:29; (g)an antibody comprising the VH polypeptide of SEQ ID NO:23 and the VLpolypeptide of SEQ ID NO:30; (h) an antibody comprising the VHpolypeptide of SEQ ID NO:69 and the VL polypeptide of SEQ ID NO:70; (i)an antibody comprising the VH polypeptide of SEQ ID NO:71 and the VLpolypeptide of SEQ ID NO:26; (j) an antibody comprising the VHpolypeptide of SEQ ID NO:71 and the VL polypeptide of SEQ ID NO:27; (k)an antibody comprising the VH polypeptide of SEQ ID NO:72 and the VLpolypeptide of SEQ ID NO:26; (l) an antibody comprising the VHpolypeptide of SEQ ID NO:72 and the VL polypeptide of SEQ ID NO:27; (m)an antibody comprising the VH polypeptide of SEQ ID NO:73 and the VLpolypeptide of SEQ ID NO:26; and (n) an antibody comprising the VHpolypeptide of SEQ ID NO:73 and the VL polypeptide of SEQ ID NO:27.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that specifically binds to human EGFR, wherein theantibody or antigen binding fragment thereof comprises: (a) animmunoglobulin heavy chain variable region comprising CDR1, CDR2, andCDR3, which, with the exception of 1, 2, or 3 conservative amino acidsubstitutions, are respectively identical to the reference heavy chainCDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2 sequence ofSEQ ID NO: 2, 4, 6, 63, or 64, and the reference heavy chain CDR3sequence of SEQ ID NO: 3 or 5; and (b) an immunoglobulin a light chainvariable region comprising CDR1, CDR2, and CDR3, which, with theexception of 1, 2, or 3 conservative amino acid substitutions, arerespectively identical to the reference light chain CDR1 sequence of SEQID NO: 10, 13, or 14, the reference light chain sequence CDR2 of SEQ IDNO:11, and the reference light chain CDR3 sequence of SEQ ID NO: 12. Inanother embodiment, the antibody comprises: (a) an immunoglobulin heavychain variable region comprising CDR1, CDR2, and CDR3, which arerespectively identical to the reference heavy chain CDR1 sequence of SEQID NO:1, the reference heavy chain CDR2 sequence of SEQ ID NO: 2, 4, 6,63, or 64, and the reference heavy chain CDR3 sequence of SEQ ID NO: 3or 5; and (b) an immunoglobulin a light chain variable region comprisingCDR1, CDR2, and CDR3, which are respectively identical to the referencelight chain CDR1 sequence of SEQ ID NO: 10, 13, or 14, the referencelight chain sequence CDR2 of SEQ ID NO:11, and the reference light chainCDR3 sequence of SEQ ID NO: 12.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that specifically binds to human EGFR, wherein theantibody or antigen binding fragment thereof comprises: (a) animmunoglobulin heavy chain variable region comprising CDR1, CDR2, andCDR3, which, with the exception of 1, 2, or 3 conservative amino acidsubstitutions, are respectively identical to the reference heavy chainCDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2 sequence ofSEQ ID NO: 7, 8, or 9, and the reference heavy chain CDR3 sequence ofSEQ ID NO: 3; and (b) an immunoglobulin a light chain variable regioncomprising CDR1, CDR2, and CDR3, which, with the exception of 1, 2, or 3conservative amino acid substitutions, are respectively identical to thereference light chain CDR1 sequence of SEQ ID NO: 15 or 16, thereference light chain CDR2 sequence of SEQ ID NO:17, and the referencelight chain CDR3 sequence of SEQ ID NO: 18. In another embodiment, theantibody comprises: (a) an immunoglobulin heavy chain variable regioncomprising CDR1, CDR2, and CDR3, which are respectively identical to thereference heavy chain CDR1 sequence of SEQ ID NO:1, the reference heavychain CDR2 sequence of SEQ ID NO: 7, 8, or 9, and the reference heavychain CDR3 sequence of SEQ ID NO: 3; and (b) an immunoglobulin a lightchain variable region comprising CDR1, CDR2, and CDR3, which arerespectively identical to the reference light chain CDR1 sequence of SEQID NO: 15 or 16, the reference light chain CDR2 sequence of SEQ IDNO:17, and the reference light chain CDR3 sequence of SEQ ID NO: 18.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that specifically binds to human EGFR, wherein theantibody or antigen binding fragment thereof comprises: (a) animmunoglobulin heavy chain variable region comprising CDR1, CDR2, andCDR3, which, with the exception of 1, 2, or 3 conservative amino acidsubstitutions, are respectively identical to the reference heavy chainCDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2 sequence ofSEQ ID NO: 65, 66, or 67, and the reference heavy chain CDR3 sequence ofSEQ ID NO: 3; and (b) an immunoglobulin a light chain variable regioncomprising CDR1, CDR2, and CDR3, which, with the exception of 1, 2, or 3conservative amino acid substitutions, are respectively identical to thereference light chain CDR1 sequence of SEQ ID NO: 68 or 13, thereference light chain sequence CDR2 of SEQ ID NO:11, and the referencelight chain CDR3 sequence of SEQ ID NO: 12. In another embodiment, theantibody comprises: (a) an immunoglobulin heavy chain variable regioncomprising CDR1, CDR2, and CDR3, which are respectively identical to thereference heavy chain CDR1 sequence of SEQ ID NO:1, the reference heavychain CDR2 sequence of SEQ ID NO: 65, 66, or 67, and the reference heavychain CDR3 sequence of SEQ ID NO: 3; and (b) an immunoglobulin a lightchain variable region comprising CDR1, CDR2, and CDR3, which arerespectively identical to the reference light chain CDR1 sequence of SEQID NO: 68 or 13, the reference light chain sequence CDR2 of SEQ IDNO:11, and the reference light chain CDR3 sequence of SEQ ID NO: 12.

In one embodiment, the antibody or antigen binding fragment of theinvention is murine, non-human, humanized, chimeric, resurfaced, orhuman. In another embodiment, the antibody or antigen binding fragmentthereof is a full length antibody. In another embodiment, the antibodyor antigen binding fragment thereof is an antigen binding fragment. Inanother embodiment, the antibody or antigen binding fragment thereofcomprises a Fab, Fab′, F(ab′)2, Fd, single chain Fv or scFv, disulfidelinked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)3,tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab,mAb2, (scFv)2, or scFv-Fc.

The invention also provides a polypeptide comprising the VH and VLsequences described herein.

In one embodiment, the invention provides an antibody or polypeptidethat binds both human and macaque EGFR with a substantially similarbinding affinity. In one embodiment, the antibody or polypeptide bindsto human and macaque EGFR with a Kd of about 1.0 to about 10 nM. Inanother embodiment, the antibody or polypeptide of binds to human andmacaque EGFR with a Kd of about 1.0 nM or better. In another embodiment,binding affinity is measured by flow cytometry, Biacore, orradioimmunoassay.

In one embodiment, the invention provides an isolated cell producing anantibody or antigen binding fragment thereof, or polypeptide describedherein.

In one embodiment, the invention provides a method of making an antibodyor antigen binding fragment thereof, or polypeptide described hereincomprising (a) culturing a cell that expresses the antibody,antigen-binding fragment thereof, or polypeptide, and (b) isolating saidantibody, antigen-binding fragment thereof, or polypeptide from saidcultured cell. In one embodiment the cell is a eukaryotic cell.

In one embodiment, the invention provides an immunoconjugate having theformula (A)-(L)-(C), wherein: (A) is an antibody or antigen bindingfragment thereof, or polypeptide described herein; (L) is a linker; and(C) is a cytotoxic agent; and wherein said linker (L) links (A) to (C).In another embodiment, the linker is selected from the group consistingof a cleavable linker, a non-cleavable linker, a hydrophilic linker, anda dicarboxylic acid based linker. In another embodiment, the linker is anon-cleavable linker. In another embodiment, the linker is selected fromthe group consisting: N-succinimidyl 4-(2-pyridyldithio)pentanoate(SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) orN-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB);N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC);N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfoSMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide). In a further embodiment, the linker isN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide).

In another embodiment, the immunoconjugate comprises a cytotoxic agentselected from the group consisting of a maytansinoid, maytansinoidanalog, doxorubicin, a modified doxorubicin, benzodiazepine, taxoid,CC-1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin,dolastatin, dolastatin analog, aristatin, tomaymycin derivative, andleptomycin derivative or a prodrug of the agent. In another embodiment,the cytotoxic agent is a maytansinoid. In a further embodiment, thecytotoxic agent isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

In one embodiment, the invention provides a pharmaceutical compositioncomprising an antibody or antigen binding fragment thereof, orpolypeptide described herein, or an immunoconjugate described herein anda pharmaceutically acceptable carrier. In another embodiment, thepharmaceutical composition comprises a second anti-cancer agent.

In one embodiment, the invention provides a diagnostic reagentcomprising an antibody or antigen binding fragment thereof, polypeptide,or immunoconjugate of the invention which is labeled. In one embodiment,the label is selected from the group consisting of a radiolabel, afluorophore, a chromophore, an imaging agent and a metal ion.

In one embodiment, the invention provides a kit comprising an antibodyor antigen binding fragment thereof, polypeptide, or immunoconjugatedescribed herein.

In one embodiment, the invention provides a method for inhibiting thegrowth of a cell expressing EGFR comprising contacting the cell with animmunoconjugate or the pharmaceutical composition described herein. Inanother embodiment, the cell is a tumor cell.

In one embodiment, the invention provides a method for treating apatient having a neoplasm comprising administering to said patient atherapeutically effective amount of an immunoconjugate or pharmaceuticalcomposition described herein. In another embodiment, the neoplasm isselected from the group consisting of: abdominal, bone, breast,digestive system, liver, pancreas, peritoneum, adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid, eye, head and neck,central nervous system, peripheral nervous system, lymphatic system,pelvic, skin, soft tissue, spleen, thoracic region, and urogenitalsystem. In another embodiment, the method comprises administering asecond anti-cancer agent to the subject. In a further embodiment, thesecond anti-cancer agent is a chemotherapeutic agent.

In one embodiment, the invention provides a method for treating a cellproliferative disorder in a patient comprising administering to saidpatient a therapeutically effective amount of an immunoconjugate orpharmaceutical composition described herein. In another embodiment, thecell proliferative disorder is selected from the group consisting of:adrenal cortex hyperplasia (Cushing's disease), congenital adrenalhyperplasia, endometrial hyperplasia, benign prostatic hyperplasia,breast hyperplasia, intimal hyperplasia, focal epithelial hyperplasia(Heck's disease), sebaceous hyperplasia, compensatory liver hyperplasia,and any other cell proliferation disease, besides neoplasia.

In one embodiment, the invention provides an isolated polynucleotidecomprising a sequence that encodes a polypeptide at least 90% identicalto a sequence selected from the group consisting of SEQ ID NOs: 39-43,77-80, and 82-84. In another embodiment, the sequence is at least 95%identical a sequence selected from the group consisting of SEQ ID NOs:39-43, 77-80, and 82-84. In another embodiment, the sequence is at least99% identical to a sequence selected from the group consisting of SEQ IDNOs: 39-43, 77-80, and 82-84.

In one embodiment, the invention provides an isolated polynucleotidewhich comprises a sequence that is at least 90% identical to SEQ ID NOs:44-50 and 81. In another embodiment, the polynucleotide comprises asequence that is at least 95% identical to SEQ ID NOs: 44-50 and 81. Inanother embodiment, the polynucleotide comprises a sequence that is atleast 99% identical to SEQ ID NOs: 44-50 and 81. In another embodiment,the invention provides an isolated polynucleotide comprising a sequenceselected from the group consisting of SEQ ID NOs:39-58 and 77-84. Inanother embodiment, the invention provides a vector comprising thepolynucleotides described herein. In another embodiment, the inventionprovides a host cell comprising the vectors described herein.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a table describing the binding affinity of the indicatedanti-EGFR antibodies to human EGFR (huEGFR) and monkey EGFR (moEGFR)antigen.

FIG. 2 is a Western blot data depicting the effect of the indicatedanti-EGFR antibodies in ligand-induced EGFR phosphorylation in MDA-MB468cells (A) and in human primary keratinocytes (B).

FIG. 3 is a Western blot data depicting the effect of the indicatedanti-EGFR antibodies on EGFR phosphorylation in MDA-MB468 cells inabsence of exogenous EGFR ligand.

FIG. 4 is a line graph depicting the binding of biotinylated TGFα (A)and EGF (B) to the A431 cells in presence of the indicated antibodies.

FIG. 5 is a line graph depicting the growth of human primarykeratinocytes in presence of the indicated antibodies at variousconcentrations.

FIG. 6 is a bar graph depicting the growth of MCF10A cells in thepresence of 10 μg/ml of the indicated antibodies.

FIG. 7 is a line graph depicting the growth of HCC827 cells (A) andNCI-H292 cells (B) in the presence of the indicated antibodies.

FIG. 8 is a line graph depicting the binding of biotinylated 528antibody (A), biotinylated cetuximab (B), and biotinylated EGFR-7antibody (C) to the MDA-MB468 cells in presence of the indicatedcompeting antibodies at the indicated concentration.

FIG. 9 is the alignment of the murine EGFR-7 variant light and heavychain variable region sequences.

FIG. 10 is tables depicting specific framework surface residue changesin resurfacing of EGFR-7 V_(L) (A) and V_(H) (B).

FIG. 11 is tables depicting specific framework surface residue changesin resurfacing of EGFR-12 V_(L) (A) and V_(H) (B).

FIG. 12 is alignment of the resurfaced sequences and murine counterpartsof EGFR-7 V_(L) (A), EGFR-7 V_(H) (B), EGFR-12 V_(L) (C) and EGFR-12V_(H) (D).

FIG. 13 is tables depicting specific framework surface residue changesin CDR grafting of EGFR-7 V_(L) (A) and V_(H) (B).

FIG. 14 is alignment of the CDR grafted sequences and the murinecounterparts of EGFR-7 V_(L) (A) and EGFR-7 V_(H) (B).

FIG. 15 shows line graphs depicting the binding competition between themurine antibody and its corresponding humanized antibody.

FIG. 16 shows line graphs depicting the ability of the murine antibodyand its corresponding humanized antibody in inhibiting tumor cellgrowth.

FIG. 17 shows a line graph depicting NK cell mediated ADCC activity ofhumanized EGFR-6 and EGFR-7R on A431 cells.

FIG. 18 depicts the binding curves of the indicated antibody and thecorresponding antibody-maytansinoid conjugate.

FIG. 19 shows line graphs depicting the cytotoxic activity of theindicated antibody and the corresponding antibody-maytansinoid conjugatein FaDu (A) and H292 (B) cell lines.

FIG. 20 shows line graphs depicting the cytotoxic activity of theindicated antibody and the corresponding antibody-maytansinoid conjugatein H226 (A) and SCC-4 (B) cell lines.

FIG. 21 shows a line graph depicting the growth of H292 tumor xenograftin mice treated with a single dose of the indicated antibodies andantibody-maytansinoid conjugates.

FIG. 22 shows a line graph depicting the growth of FaDu tumor xenograftin mice treated with a single dose of the indicated antibodies andantibody-maytansinoid conjugates.

FIG. 23 shows line graphs depicting the capacity of the indicatedantibodies and conjugates in inhibiting the growth of human primarykeratinocytes (A) and H292 tumor cells (B).

FIG. 24 shows bar graphs depicting the amount of CXCL8, CXCL10, CCL5produced by the human primary keratinocytes in presence of the indicatedantibodies and antibody-maytansinoid-conjugates.

FIG. 25 shows a line graph depicting the growth of H292 tumor xenograftin mice treated with a single 3 mg/kg dose of either the huEGFR-7Rantibody or the huEGFR-7R-SMCC-DM1, or huEGFR-7R-PEG-MAL-DM1antibody-maytansinoid conjugates.

FIG. 26 shows a line graph depicting the growth of HSC2 tumor xenograftin mice treated with a single 5 mg/kg dose of either the huEGFR-7Rantibody or the huEGFR-7R-SMCC-DM1, or huEGFR-7R-PEG-MAL-DM1antibody-maytansinoid conjugates.

FIG. 27 shows a line graph depicting the growth of FaDu tumor xenograftin mice treated with a single 5 mg/kg dose of either the huEGFR-7Rantibody or the huEGFR-7R-SMCC-DM1, or huEGFR-7R-PEG-MAL-DM1antibody-maytansinoid conjugates.

FIG. 28 shows alignment of human and murine EGFR extracellular domain(ECD) sequences.

FIG. 29 shows alignment of sequences of huEGFRdIII, muEGFRdIII andchEGFRdIII.

FIG. 30 shows a line graph depicting the binding of huEGFR-7R antibodyto the huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 31 shows a line graph depicting the binding of huEGFR-6 antibody tothe huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 32 shows a line graph depicting the binding of muEGFR-7 antibody tothe huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 33 shows a line graph depicting the binding of muEGFR-6 antibody tothe huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 34 shows a line graph depicting the binding of muEGFR-12 antibodyto the huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 35 shows a line graph depicting the binding of muEGFR-13 antibodyto the huEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

FIG. 36 shows a line graph depicting the binding of cetuximab to thehuEGFR, huEGFRdIII, muEGFRdIII and chEGFRdIII.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new class of EGFR binding moleculeswhich partially inhibit EGFR signaling, have no effect on EGFR-positivenormal epithelial cells, including primary keratinocytes, but are highlycytotoxic to EGFR-overexpressing tumor cells. Further, immunoconjugatesof anti-EGFR antibodies potentiate the anti-tumor activity of theantibodies.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, “epidermal growth factor receptor” or “EGFR” refers tothe mature, tyrosine kinase cell surface receptor. The term “solubleEGFR” or “sEGFR” refers to a portion of EGFR containing theextracellular, ligand-binding domain of EGFR. More specifically, sEGFRcontains amino acids 1-619 of mature EGFR (Ullrich et al., HumanEpidermal Growth Factor cDNA Sequence and Aberrant Expression of theAmplified Gene in A-431 Epidermoid Carcinoma Cells, Nature, Vol. 309,418-25 (1986)).

The phrase “EGFR mediated cancer” refers to a cancer characterized byepithelial tumors in which EGFR is abnormally activated to levelsgreater than in normal, corresponding epithelial tissue. These greaterlevels of EGFR activity promote tumor growth in many types of cancer.Such cancers include, but are not limited to, non-small cell lungcancer, breast cancer, colorectal cancer, head and neck cancers, andprostate cancer. Abnormal activation of EGFR can arise fromoverexpression of the receptor, gene amplification, activatingmutations, overexpression of receptor ligands, and/or loss of regulatorsof EGFR activity.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as EGFR. Insome embodiments, blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. The biological activity can be reduced by 10%, 20%, 30%, 50%,70%, 80%, 90%, 95%, or even 100%.

The phrase “ability to inhibit EGFR activation” with respect to anantibody as used herein, is intended to refer to an antibody whosebinding to EGFR results in inhibition of human EGFR activation and thebiological activity of human EGFR that occurs upon activation of thereceptor. Measuring one or more indicators of EGFR biological activityas determined using either a cell proliferation assay, an apoptosisassay, a receptor binding assay, a receptor phosphorylation assay, or amouse tumor model (see Examples) can assess an antibody's ability toinhibit EGFR activation.

The term “anti-EGFR antibody” or “an antibody that binds to EGFR” refersto an antibody that is capable of binding EGFR with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting EGFR. Several anti-EGFR antibodies are known in theart. For example, cetuximab (Ab 225) and 528 Ab are described in U.S.Pat. No. 4,943,533, which is herein incorporated by reference.

The extent of binding of an anti-EGFR antibody to an unrelated, non-EGFRprotein can be less than about 10% of the binding of the antibody toEGFR as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that binds to EGFR has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM.

As used herein, the term “epithelial toxicity” refers to an abnormalityor dysfunction of the epithelium, and can be manifested in a patientbeing treated for cancer by administration of an EGFR inhibitor by oneor more symptoms or conditions selected from skin rash, diarrhea,corneal thinning, hair atrophy or loss, hair follicle dysplasia,degeneration, necrosis or inflammation, interfollicular epidermalhyperplasia, or a failure to heal or a delayed healing after injury,among other symptoms. In one embodiment, the epithelial toxicity ismanifested as a skin toxicity such as acneform or macro-papular rash.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better”, the antibody's affinity for theantigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values can be less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10% as a function ofthe value for the reference/comparator antibody. The difference betweentwo “substantially similar binding affinities” is generally less thanabout 10% as a function of the value for the reference/comparatorantibody.

“Ligand-independent binding” as used herein denotes the ability of theEGFR binding agents to bind an epitope on human EGFR in the absence ofligand interaction with EGFR.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is linked to a cell binding agent(i.e., an anti-EGFR antibody or fragment thereof) and is defined by ageneric formula: C-L-A, wherein C=cytotoxin, L=linker, and A=cellbinding agent or anti-EGFR antibody or antibody fragment.Immunoconjugates can also be defined by the generic formula in reverseorder: A-L-C.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a maytansinoid, to a cell-binding agent such asan anti-EGFR antibody or a fragment thereof in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude charged linkers, and hydrophilic forms thereof as describedherein and know in the art.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. “Tumor” and “neoplasm” refer to one or more cells that resultfrom excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.Examples of “cancer” or “tumorigenic” diseases which can be treatedand/or prevented include neoplasms of the abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. The formulation can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent or stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentor stop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. See the definition herein of “treating”. To the extent the drugcan prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result. Typically but notnecessarily, since a prophylactic dose is used in subjects prior to orat an earlier stage of disease, the prophylactically effective amountwill be less than the therapeutically effective amount.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Chemotherapeuticagents include, for example, antagonists of CD20 such as Rituximab andcyclophosphamide, doxorubicin, vincristine, predinisone, fludarabine,etoposide, methotrexate, lenalidomide, chlorambucil, bentamustine and/ormodified versions of such chemotherapeutics.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars can be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or can be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping group moieties of from 1 to 20 carbon atoms. Otherhydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages can be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

The term “vector” means a construct, which is capable of delivering, andoptionally expressing, one or more gene(s) or sequence(s) of interest ina host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTcan be used as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Identity can exist over a region of the sequences that is at least about10, about 20, about 40-60 residues in length or any integral valuetherebetween, and can be over a longer region than 60-80 residues, forexample, at least about 90-100 residues, and in some embodiments, thesequences are substantially identical over the full length of thesequences being compared, such as the coding region of a nucleotidesequence for example.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In someembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the EGFR to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

II. EGFR Binding Agents

Tumors often overexpress growth factor receptors that bind variousligands ligand and facilitate unrestricted tumor growth. One example ofsuch growth factor receptors are the receptors of the Epidermal GrowthFactor Receptor (EGFR) protein family.

Signal transduction through members of the Epidermal Growth FactorReceptor (EGFR) protein family is dependent upon the formation ofhomodimers or heterodimers triggered by the binding of ligand. Thisreceptor family is comprised of four membrane-bound proteins: EGFR,HER2/neu, HER3 and HER4. Overexpression of these proteins has beencorrelated with a poor prognosis in a number of types of cancer,including, but not limited to, breast, colon, ovarian, endometrial,gastric, pancreatic, prostate and salivary gland cancers. While a numberof groups have developed strategies to target individual members of theEGFR protein family (e.g., HER2/neu or EGFR) to inhibit tumor growth,none of the treatments has been proven to ultimately cure these forms ofcancer.

In accordance with this invention novel agents (e.g. antibodies) areprovided that specifically bind to human EGFR. These novel agentspartially inhibit EGFR signaling and have no effect on EGFR-positivenormal epithelial cells, including primary keratinocytes. However, theseagents are highly cytotoxic to EGFR-overexpressing tumor cells.

Thus, present invention provides agents that specifically bind humanEGFR. These agents are referred to herein as “EGFR binding agents.” Incertain embodiments, the EGFR binding agents are antibodies,immunoconjugates or polypeptides. In some embodiments, the EGFR bindingagents are humanized antibodies.

In certain embodiments, the EGFR-binding agents have one or more of thefollowing effects: inhibit proliferation of tumor cells, reduce thetumorigenicity of a tumor by reducing the frequency of cancer stem cellsin the tumor, inhibit tumor growth, increase survival, trigger celldeath of tumor cells, differentiate tumorigenic cells to anon-tumorigenic state, or prevent metastasis of tumor cells.

In some embodiments, the EGFR-binding agents are capable of reducingtumor volume. The ability of a EGFR-binding agent to reduce tumor volumecan be assessed, for example, by measuring a % T/C value, which is themedian tumor volume of treated subjects divided by the median tumorvolume of the control subjects. In some embodiments, treatment with aEGFR-binding agent results in a % T/C value that is less than about 55%,less than about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, or less than about 5%.

In certain embodiments, immunoconjugates or other agents thatspecifically bind human EGFR trigger cell death via a cytotoxic agent.For example, in certain embodiments, an antibody to a human EGFRantibody is conjugated to a maytansinoid that is activated in tumorcells expressing the EGFR by protein internalization. In certainalternative embodiments, the agent or antibody is not conjugated.

In certain embodiments, the EGFR-binding agents are capable ofinhibiting tumor growth. In certain embodiments, the EGFR-binding agentsare capable of inhibiting tumor growth in vivo (e.g., in a xenograftmouse model and/or in a human having cancer).

The EGFR-binding agents include EGFR antibodies EGFR-6, EGFR-7, andEGFR-12 and fragments, variants and derivatives thereof. TheEGFR-binding agents also include EGFR-binding agents that specificallybind to the same EGFR epitope as the antibodies EGFR-6, EGFR-7, andEGFR-12. The EGFR-binding agents also include EGFR-binding agents thatcompetitively inhibit the antibodies EGFR-6, EGFR-7, and EGFR-12.

The EGFR-binding agents also include EGFR-binding agents that comprisethe heavy and light chain CDR sequences of EGFR-6, EGFR-7, and EGFR-12.The CDR sequences of EGFR-7 and EGFR-12, both murine and humanized aredescribed in Tables 1 and 2 below.

TABLE 1 Variable heavy chain CDR amino acid sequences Antibody VH-CDR1VH-CDR2 VH-CDR3 EGFR-7 TSYWMQ TIYPGDGDTT YDAPGYAMDY (SEQ ID NO: 1)(SEQ ID NO: 2) (SEQ ID NO: 3) EGFR-7 TSYWMQ Xaa₁Xaa₂YPGDGDXaa₃Xaa₄;YDAPGYXaa₁MDY variant (SEQ ID NO: 1) Xaa₁ = T or A Xaa₁ = A or Tcomposite Xaa₂ = I or L (SEQ ID NO: 5) sequences Xaa₃ = T or A Xaa₄ =T, R, or S (SEQ ID NO: 4) EGFR-7 Xaa₁Xaa₂YPGDGDXaa₃Xaa₄Xaa₅QKFXaa₆GKabat HC Xaa₁ = T or A CDR2 Xaa₂ = I or L Xaa₃ = T or A Xaa₄ =T, R, or S Xaa₅ = Y, T, or I Xaa₆ = Q or K (SEQ ID NO: 6) EGFR-12 TSYWMQTIYPGDGDTR YDAPGYAMDY (SEQ ID NO: 1) (SEQ ID NO: 7) (SEQ ID NO: 3)Kabat HC TIYPGDGDTRYIQKFKG (murine) (SEQ ID NO: 8) Kabat HCTIYPGDGDTRYIQKFQG (resurfaced) (SEQ ID NO: 9) EGFR-6 TSYWMQ ALYPGDGDARYDAPGYAMDY (SEQ ID NO: 1) (SEQ ID NO: 65) (SEQ ID NO: 3) Kabat HCALYPGDGDARYTQKFKG (murine) (SEQ ID NO: 66) Kabat HC ALYPGDGDARYTQKFQG(resurfaced) (SEQ ID NO: 67)

TABLE 2 Variable light chain CDR amino acid sequences Antibody VL-CDR1VL-CDR2 VL-CDR3 EGFR-7 KASQDINNYLA YTSTLHP LQYDNLLYT (murine and(SEQ ID NO: 11) (SEQ ID NO: 12) resurfaced v1.01) (SEQ ID NO: 10)RASQDINNYLA (resurfaced v1.0 and CDR grafted) (SEQ ID NO: 13) EGFR-7Xaa₁ASQDINNYXaa₂A YTSTLHP LQYDNLLYT variant Xaa₁ = K or R(SEQ ID NO: 11) (SEQ ID NO: 12) composite Xaa₂ = L or I sequences(SEQ ID NO: 14) EGFR-12 RASKSISKYLA (murine and resurfaced v1.01)(SEQ ID NO: 15) RASQSISRYLA SGSTLQS QQHNEYPWT (resurfaced(SEQ ID NO: 17) (SEQ ID NO: 18) v1.0) (SEQ ID NO: 16) EGFR-6 KASQDINNYIAYTSTLHP LQYDNLLYT (murine) (SEQ ID NO: 68) (SEQ ID NO: 11)(SEQ ID NO: 12) (resurfaced) RASQDINNYLA (SEQ ID NO: 13)

The EGFR binding molecules can be antibodies or antigen bindingfragments that specifically bind to EGFR that comprise the CDRs ofEGFR-6, EGFR-7, and EGFR-12 with up to four (i.e. 0, 1, 2, 3, or 4)conservative amino acid substitutions per CDR.

Polypeptides can comprise one of the individual variable light chains orvariable heavy chains described herein. Antibodies and polypeptides canalso comprise both a variable light chain and a variable heavy chain.The variable light chain and variable heavy chain sequences of murineand humanized EGFR-7 and EGFR-12 antibodies are provided in Tables 3 and4 below.

TABLE 3 Variable heavy chain amino acid sequences AntibodyVH Amino Acid Sequence muEGFR-7QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYPG V_(H)DGDTTYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARYDAPGYAMD YWGQGTSVTVSS(SEQ ID NO: 19) muEGFR-12QVQLQQSGTELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYPG V_(H)DGDTRYIQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARYDAPGYAMD YWGQGTSVTVSS(SEQ ID NO: 20) huEGFR-7QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYP V_(H)GDGDTTYTQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAM DYWGQGTLVTVSS(SEQ ID NO: 21) huEGFR-7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQWVRQAPGQGLEWMGTIY V_(H)_CDRPGDGDTTYTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYDAPGYA graftedMDYWGQGTLVTVSS (SEQ ID NO: 22) huEGFR-12QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYP V_(H)GDGDTRYIQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAM DYWGQGTLVTVSS(SEQ ID NO: 23) muEGFR-6QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGALYP V_(H)GDGDARYTQKFKGKATLTADRSSSTAYMQLSSLASEDSAVYYCARYDAPGYAM DYWGQGTSVTVAS(SEQ ID NO: 69) huEGFR-6QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGALYP V_(H) v1.0GDGDARYTQKFQGKATLTADTSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAM DYWGQGTLVTVSS(SEQ ID NO: 71) huEGFR-6QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGALY V_(H) v1.11PGDGDARYTQKFQGKATLTADTSSSTAYMQLSSLRSEDSAVYYCARYDAPGYA MDYWGQGTLVTVSS(SEQ ID NO: 72) huEGFR-7QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGTIYP V_(H) v1.11GDGDTTYTQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAM DYWGQGTLVTVSS(SEQ ID NO: 73)

TABLE 4 Variable light chain amino acid sequences AntibodyVL Amino Acid Sequence muEGFR-DIQMTQSPSSLSASLGGKVTITCKASQDINNYLAWYQHKPGKGPRLLIHYTSTLHP 7 V_(L)GIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLYTFGGGTKLEIKR (SEQ ID NO: 24)muEGFR- DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGI12 V_(L) PSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIKR(SEQ ID NO: 25) huEGFR-7DIQMTQSPSSLSASVGDRVTITCRASQDINNYLAWYQHKPGKGPKLLIHYTSTLHP V_(L) v1.0GIPSRFSGSGSGRDYSFSISSLEPEDIATYYCLQYDNLLYTFGQGTKLEIKR (SEQ ID NO: 26)huEGFR-7 DIQMTQSPSSLSASVGDRVTITCKASQDINNYLAWYQHKPGKGPKLLIHYTSTLHPV_(L) v1.01 GIPSRFSGSGSGRDYSFSISSLEPEDIATYYCLQYDNLLYTFGQGTKLEIKR(SEQ ID NO: 27) huEGFR-7DIQMTQSPSSLSASVGDRVTITCRASQDINNYLAWYQQKPGKAPKLLIYYTSTLHP V_(L)_CDRGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLLYTFGQGTKVEIKR grafted(SEQ ID NO: 28) huEGFR-DVQITQSPSSLAASVGERITINCRASQSISRYLAWYQEKPGKTNKLLIYSGSTLQSG 12 V_(L) IPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGQGTKLEIKR v1.0 (SEQ ID NO: 29)huEGFR- DVQITQSPSSLAASVGERITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSG12 V_(L) IPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGQGTKLEIKR v1.01(SEQ ID NO: 30) muEGFR6DIQMTQSPSSLSASLGGKVTITCKASQDINNYIAWYQHKPGKGPRLLIHYTSTLHP V_(L)GIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLYTFGGGTKLEIKR (SEQ ID NO: 70)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:19-23, 69, and 71-76;and/or (b) a polypeptide having at least about 90% sequence identity toSEQ ID NOs:24-30 and 70. In certain embodiments, the polypeptidecomprises a polypeptide having at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NOs:19-30 and 69-76. Thus, in certain embodiments,the polypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NOs: 19-23, 69, and 71-76, and/or (b) apolypeptide having at least about 95% sequence identity to SEQ IDNOs:24-30 and 70. In certain embodiments, the polypeptide comprises (a)a polypeptide having the amino acid sequence of SEQ ID NOs: 19-23, 69,and 71-76; and/or (b) a polypeptide having the amino acid sequence ofSEQ ID NOs:24-30 and 70. In certain embodiments, the polypeptide is anantibody and/or the polypeptide specifically binds EGFR. In certainembodiments, the polypeptide is a murine, chimeric, or humanizedantibody that specifically binds EGFR. In certain embodiments, thepolypeptide having a certain percentage of sequence identity to SEQ IDNOs: 19-30 and 69-76 differs from SEQ ID NOs: 19-30 and 69-76 byconservative amino acid substitutions only.

TABLE 5 Full-length heavy chain and light chain amino acid sequencesAntibody VH Amino Acid Sequence huEGFR-7QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYPGD HCGDTTYTQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31)huEGFR-7 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMQWVRQAPGQGLEWMGTIYPG HC_CDRDGDTTYTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYDAPGYAMDYW graftedGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32)huEGFR-7 DIQMTQSPSSLSASVGDRVTITCRASQDINNYLAWYQHKPGKGPKLLIHYTSTLHPGILCv1.0 PSRFSGSGSGRDYSFSISSLEPEDIATYYCLQYDNLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33) huEGFR-7DIQMTQSPSSLSASVGDRVTITCKASQDINNYLAWYQHKPGKGPKLLIHYTSTLHPGI LCv1.01PSRFSGSGSGRDYSFSISSLEPEDIATYYCLQYDNLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34) huEGFR-7DIQMTQSPSSLSASVGDRVTITCRASQDINNYLAWYQQKPGKAPKLLIYYTSTLHPG LC_CDRVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLLYTFGQGTKVEIKRTVAAPSV graftedFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) huEGFR-12QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGTIYPGD HCGDTRYIQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(SEQ ID NO: 36) huEGFR-12DVQITQSPSSLAASVGERITINCRASQSISRYLAWYQEKPGKTNKLLIYSGSTLQSGI LCv.1.0PSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 37) huEGFR-12DVQITQSPSSLAASVGERITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGI LCv.1.01PSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 38) huEGFR-6QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLECIGALYPG HCv1.0DGDARYTQKFQGKATLTADTSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG(SEQ ID NO: 74) huEGFR-6QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGALYP HCv1.11GDGDARYTQKFQGKATLTADTSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG(SEQ ID NO: 75) huEGFR-7QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGTIYPG HCv1.11DGDTTYTQKFQGKATLTADKSSSTAYMQLSSLRSEDSAVYYCARYDAPGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG(SEQ ID NO: 76)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:31, 32, and 36;and/or (b) a polypeptide having at least about 90% sequence identity toSEQ ID NOs:33-35, 37, and 38. In certain embodiments, the polypeptidecomprises a polypeptide having at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NOs:31-38. Thus, in certain embodiments, thepolypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NOs: 31, 32, and 36, and/or (b) apolypeptide having at least about 95% sequence identity to SEQ ID NOs:33-35, 37, and 38. In certain embodiments, the polypeptide comprises (a)a polypeptide having the amino acid sequence of SEQ ID NOs: 31, 32, and36; and/or (b) a polypeptide having the amino acid sequence of SEQ IDNOs: 33-35, 37, and 38. In certain embodiments, the polypeptide is anantibody and/or the polypeptide specifically binds EGFR. In certainembodiments, the polypeptide is a humanized antibody that specificallybinds EGFR. In certain embodiments, the polypeptide having a certainpercentage of sequence identity to SEQ ID NOs:31-38 differs from SEQ IDNOs:31-38 by conservative amino acid substitutions only.

In certain embodiments, the EGFR antibody is the antibody produced froma hybridoma selected from the group consisting of ATCC DepositDesignation PTA-11331 (EGFR-6), deposited with the ATCC on Oct. 6, 2010,ATCC Deposit Designation PTA-11332 (EGFR-7), deposited with the ATCC onOct. 6, 2010, and ATCC Deposit Designation PTA-11333 (EGFR-12),deposited with the ATCC on Oct. 6, 2010. In certain embodiments, theantibody comprises the VH-CDRs and the VL-CDRS of the antibody producedfrom a hybridoma selected from the group consisting of PTA-11331,PTA-11332, and PTA-11333.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human EGFR is ahumanized antibody. In certain embodiments, such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject. Humanizedantibodies can be produced using various techniques known in the art. Incertain alternative embodiments, the antibody to EGFR is a humanantibody.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, e.g., Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., 1992,Bio/Technology 10:779-783, incorporated by reference in its entirety)are known in the art and can be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a EGFR. Bispecific antibodies are antibodies that are capableof specifically recognizing and binding at least two different epitopes.The different epitopes can either be within the same molecule (e.g. thesame EGFR) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a EGFR as well as, forexample, 1) an effector molecule on a leukocyte such as a T-cellreceptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) acytotoxic agent as described in detail below.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodimentsthe antibodies to EGFR are multispecific.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to EGFR (see U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (Huse, et al., Science 246:1275-1281 (1989))to allow rapid and effective identification of monoclonal Fab fragmentswith the desired specificity for EGFR, or derivatives, fragments,analogs or homologs thereof. Antibody fragments can be produced bytechniques in the art including, but not limited to: (a) a F(ab′)2fragment produced by pepsin digestion of an antibody molecule; (b) a Fabfragment generated by reducing the disulfide bridges of an F(ab′)2fragment, (c) a Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman EGFR. In this regard, the variable region can comprise or bederived from any type of mammal that can be induced to mount a humoralresponse and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and possibly from an antibody from adifferent species. It is not always necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, insome cases it is only necessary to transfer those residues that arenecessary to maintain the activity of the antigen binding site. Giventhe explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein can comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2 or CH3) and/or to the lightchain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g. 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the EGFR-binding antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain canreduce Fc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases, it can be that constantregion modifications, consistent with this invention, moderatecomplement binding and thus reduce the serum half-life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to eliminate disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. Similarly,modifications to the constant region in accordance with this inventioncan easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain embodiments, a EGFR-binding agent that is an antibody doesnot have one or more effector functions. For instance, in someembodiments, the antibody has no antibody-dependent cellularcytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity(CDC) activity. In certain embodiments, the antibody does not bind to anFc receptor and/or complement factors. In certain embodiments, theantibody has no effector function.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it can bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain can be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it can be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it can be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it can be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human EGFR. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against EGFR protein. Such mutantsinclude deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biologicalhalf-life or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human EGFR. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-EGFR antibody, or fragment thereof,operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it can include an N-terminalmethionine residue. This residue can optionally be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a EGFR-binding polypeptide orantibody (or a EGFR protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a EGFR-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNo. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the EGFR-binding agent is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gillet al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety. In certainembodiments, phage display technology has been used to identify/producethe EGFR-binding polypeptide. In certain embodiments, the polypeptidecomprises a protein scaffold of a type selected from the groupconsisting of protein A, a lipocalin, a fibronectin domain, an ankyrinconsensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinEGFR-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., J. Comb. Chem., 10:345-354 (2008), Dolle et al, J. Comb.Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8:1383-404(2001), each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. No. 5,270,163, U.S. Pat.No. 5,683,867, U.S. Pat. No. 5,763,595, U.S. Pat. No. 6,344,321, U.S.Pat. No. 7,368,236, U.S. Pat. No. 5,582,981, U.S. Pat. No. 5,756,291,U.S. Pat. No. 5,840,867, U.S. Pat. No. 7,312,325, U.S. Pat. No.7,329,742, International Patent Publication No. WO 02/077262,International Patent Publication No. WO 03/070984, U.S. PatentApplication Publication No. 2005/0239134, U.S. Patent ApplicationPublication No. 2005/0124565, and U.S. Patent Application PublicationNo. 2008/0227735, each of which is incorporated by reference herein inits entirety.

III. Immunoconjugates

The present invention is also directed to conjugates (also referred toherein as immunoconjugates), comprising the anti-EGFR antibodies,antibody fragments, and their functional equivalents as disclosedherein, linked or conjugated to a drug or prodrug. Suitable drugs orprodrugs are known in the art. The drugs or prodrugs can be cytotoxicagents. The cytotoxic agent used in the cytotoxic conjugate of thepresent invention can be any compound that results in the death of acell, or induces cell death, or in some manner decreases cell viability,and includes, for example, maytansinoids and maytansinoid analogs. Othersuitable cytotoxic agents are for example benzodiazepines, taxoids,CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs,enediynes, such as calicheamicins, dolastatin and dolastatin analogsincluding auristatins, tomaymycin derivatives, leptomycin derivaties,methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil andmorpholino doxorubicin.

Such conjugates can be prepared by using a linking group in order tolink a drug or prodrug to the antibody or functional equivalent.Suitable linking groups are well known in the art and include, forexample, disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.

The drug or prodrug can, for example, be linked to the anti-EGFRantibody or fragment thereof through a disulfide bond. The linkermolecule or crosslinking agent comprises a reactive chemical group thatcan react with the anti-EGFR antibody or fragment thereof. The reactivechemical groups for reaction with the cell-binding agent can beN-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally thelinker molecule comprises a reactive chemical group, which can be adithiopyridyl group that can react with the drug to form a disulfidebond. Linker molecules include, for example, N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al.,Biochem. J., 173: 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate(sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example,the antibody or cell binding agent can be modified with crosslinkingreagents and the antibody or cell binding agent containing free orprotected thiol groups thus derived is then reacted with a disulfide- orthiol-containing maytansinoid to produce conjugates. The conjugates canbe purified by chromatography, including but not limited to HPLC,size-exclusion, adsorption, ion exchange and affinity capture, dialysisor tangential flow filtration.

In another aspect of the present invention, the anti-EGFR antibody islinked to cytotoxic drugs via disulfide bonds and a polyethylene glycolspacer in enhancing the potency, solubility or the efficacy of theimmunoconjugate. Such cleavable hydrophilic linkers are described inWO2009/0134976. The additional benefit of this linker design is thedesired high monomer ratio and the minimal aggregation of theantibody-drug conjugate. Specifically contemplated in this aspect areconjugates of cell-binding agents and drugs linked via disulfide group(—S—S—) bearing polyethylene glycol spacers ((CH₂CH₂O)_(n=1-14)) with anarrow range of drug load of 2-8 are described that show relatively highpotent biological activity toward cancer cells and have the desiredbiochemical properties of high conjugation yield and high monomer ratiowith minimal protein aggregation.

Specifically contemplated in this aspect is an anti-EGFR antibody drugconjugate of formula (I) or a conjugate of formula (I′):

CB—[X₁—(—CH₂—CH₂O—)_(n)—Y-D]_(m)  (I)

[D-Y—(—CH₂—CH₂O—)_(n)—X₁]_(m)—CB  (I′)

wherein:

CB represents an anti-EGFR antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit attachedto the cell-binding agent via a thioether bond, an amide bond, acarbamate bond, or an ether bond;

Y represents an aliphatic, an aromatic or a heterocyclic unit attachedto the drug via a disulfide bond;

1 is 0 or 1;

m is an integer from 2 to 8; and

n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer form 2 to 8. Alternatively, asdisclosed in, for example, U.S. Pat. Nos. 6,441,163 and 7,368,565, thedrug can be first modified to introduce a reactive ester suitable toreact with a cell-binding agent. Reaction of these drugs containing anactivated linker moiety with a cell-binding agent provides anothermethod of producing a cell-binding agent drug conjugate. Maytansinoidscan also be linked to anti-EGFR antibody or fragment using PEG linkinggroups, as set forth for example in U.S. Pat. No. 6,716,821. These PEGnon-cleavable linking groups are soluble both in water and innon-aqueous solvents, and can be used to join one or more cytotoxicagents to a cell binding agent. Exemplary PEG linking groups includeheterobifunctional PEG linkers that react with cytotoxic agents and cellbinding agents at opposite ends of the linkers through a functionalsulfhydryl or disulfide group at one end, and an active ester at theother end. As a general example of the synthesis of a cytotoxicconjugate using a PEG linking group, reference is again made to U.S.Pat. No. 6,716,821 which is incorporated entirely by reference herein.Synthesis begins with the reaction of one or more cytotoxic agentsbearing a reactive PEG moiety with a cell-binding agent, resulting indisplacement of the terminal active ester of each reactive PEG moiety byan amino acid residue of the cell binding agent, to yield a cytotoxicconjugate comprising one or more cytotoxic agents covalently bonded to acell binding agent through a PEG linking group. Alternatively, the cellbinding can be modified with the bifunctional PEG crosslinker tointroduce a reactive disulfide moiety (such as a pyridyldisulfide),which can then be treated with a thiol-containing maytansinoid toprovide a conjugate. In another method, the cell binding can be modifiedwith the bifunctional PEG crosslinker to introduce a thiol moiety whichcan then can be treated with a reactive disulfide-containingmaytansinoid (such as a pyridyldisulfide), to provide a conjugate.

Antibody-maytansinoid conjugates with non-cleavable links can also beprepared. Such crosslinkers are described in the art (see US PublicationNo. 20050169933) and include but are not limited to, N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC). In some embodiments,the antibody is modified with crosslinking reagents such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS orsuccinimidyl-iodoacetate, as described in the literature, to introduce1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101:395-399(1979); Hashida et al, J. Applied Biochem., 56-63 (1984); and Liu et al,Biochem., 18:690-697 (1979)). The modified antibody is then reacted withthe thiol-containing maytansinoid derivative to produce a conjugate. Theconjugate can be purified by gel filtration through a Sephadex G25column or by dialysis or tangential flow filtration. The modifiedantibodies are treated with the thiol-containing maytansinoid (1 to 2molar equivalent/maleimido group) and antibody-maytansinoid conjugatesare purified by gel filtration through a Sephadex G-25 column,chromatography on a ceramic hydroxyapatite column, dialysis ortangential flow filtration or a combination of methods thereof.Typically, an average of 1-10 maytansinoids per antibody are linked. Onemethod is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again conjugates with 1 to 10 drug molecules per antibody moleculeresult. Maytansinoid conjugates of antibodies, antibody fragments, andother proteins are made in the same way.

In another aspect of the invention, the EGFR antibody is linked to thedrug via a non-cleavable bond through the intermediacy of a PEG spacer.Suitable crosslinking reagents comprising hydrophilic PEG chains thatform linkers between a drug and the anti-EGFR antibody or fragment arealso well known in the art, or are commercially available (for examplefrom Quanta Biodesign, Powell, Ohio). Suitable PEG-containingcrosslinkers can also be synthesized from commercially available PEGsthemselves using standard synthetic chemistry techniques known to oneskilled in the art. The drugs can be reacted with bifunctionalPEG-containing cross linkers to give compounds of the following formula,Z—X₁—(—CH₂—CH₂—O—)_(n)—Y_(p)-D, by methods described in detail in USPatent Publication 20090274713 and in WO2009/0134976, which can thenreact with the cell binding agent to provide a conjugate. Alternatively,the cell binding can be modified with the bifunctional PEG crosslinkerto introduce a thiol-reactive group (such as a maleimide orhaloacetamide) which can then be treated with a thiol-containingmaytansinoid to provide a conjugate. In another method, the cell bindingcan be modified with the bifunctional PEG crosslinker to introduce athiol moiety which can then be treated with a thiol-reactivemaytansinoid (such as a maytansinoid bearing a maleimide orhaloacetamide), to provide a conjugate.

Accordingly, another aspect of the present invention is an anti-EGFRantibody drug conjugate of formula (II) or of formula (II'):

CB—[X₁—(—CH₂—CH₂—O—)_(n)—Y_(p)-D]_(m)  (II)

[D—Y_(p)—(—CH₂—CH₂—O—)_(n)—X₁]_(m)—CB  (II′)

wherein, CB represents an anti-EGFR antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit bonded tothe cell-binding agent via a thioether bond, an amide bond, a carbamatebond, or an ether bond;

Y represents an aliphatic, an aromatic, or a heterocyclic unit bonded tothe drug via a covalent bond selected from the group consisting of athioether bond, an amide bond, a carbamate bond, an ether bond, an aminebond, a carbon-carbon bond and a hydrazone bond;

l is 0 or 1;

p is 0 or 1;

m is an integer from 2 to 15; and

n is an integer from 1 to 2000.

In some embodiments, m is an integer from 2 to 8; and

In some embodiments, n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer from 2 to 8. Examples of suitablePEG-containing linkers include linkers having an N-succinimidyl ester orN-sulfosuccinimidyl ester moiety for reaction with the anti-EGFRantibody or fragment thereof, as well as a maleimido- orhaloacetyl-based moiety for reaction with the compound. A PEG spacer canbe incorporated into any crosslinker known in the art by the methodsdescribed herein.

Many of the linkers disclosed herein are described in detail in U.S.Patent Publication Nos. 20050169933 and 20090274713, and inWO2009/0134976; the contents of which are entirely incorporated hereinby reference.

The present invention includes aspects wherein about 2 to about 8 drugmolecules (“drug load”), for example, maytansinoid, are linked to ananti-EGFR antibody or fragment thereof, the anti-tumor effect of theconjugate is much more efficacious as compared to a drug load of alesser or higher number of drugs linked to the same cell binding agent.“Drug load”, as used herein, refers to the number of drug molecules(e.g., a maytansinoid) that can be attached to a cell binding agent(e.g., an anti-EGFR antibody or fragment thereof). In one aspect thenumber of drug molecules that can be attached to a cell binding agentcan average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1). N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1)and N^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl) maytansine(DM4) can be used.

The anti-EGFR antibody or fragment thereof can be modified by reacting abifunctional crosslinking reagent with the anti-EGFR antibody orfragment thereof, thereby resulting in the covalent attachment of alinker molecule to the anti-EGFR antibody or fragment thereof. As usedherein, a “bifunctional crosslinking reagent” is any chemical moietythat covalently links a cell-binding agent to a drug, such as the drugsdescribed herein. In another method, a portion of the linking moiety isprovided by the drug. In this respect, the drug comprises a linkingmoiety that is part of a larger linker molecule that is used to join thecell-binding agent to the drug. For example, to form the maytansinoidDM1, the side chain at the C-3 hydroxyl group of maytansine is modifiedto have a free sulfhydryl group (SH). This thiolated form of maytansinecan react with a modified cell-binding agent to form a conjugate.Therefore, the final linker is assembled from two components, one ofwhich is provided by the crosslinking reagent, while the other isprovided by the side chain from DM1.

The drug molecules can also be linked to the antibody molecules throughan intermediary carrier molecule such as serum albumin.

As used herein, the expression “linked to a cell-binding agent” or“linked to an anti-EGFR antibody or fragment” refers to the conjugatemolecule comprising at least one drug derivative bound to a cell-bindingagent anti-EGFR antibody or fragment via a suitable linking group, or aprecursor thereof. One linking group is SMCC.

In certain embodiments, cytotoxic agents useful in the present inventionare maytansinoids and maytansinoid analogs. Examples of suitablemaytansinoids include esters of maytansinol and maytansinol analogs.Included are any drugs that inhibit microtubule formation and that arehighly toxic to mammalian cells, as are maytansinol and maytansinolanalogs.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219;4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598;4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410;7,276,497 and 7,473,796.

In a certain embodiment, the immunoconjugates of the invention utilizethe thiol-containing maytansinoid (DM1), formally termedN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, as thecytotoxic agent. DM1 is represented by the following structural formula(III):

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoidN^(2′)-deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine(e.g., DM4) as the cytotoxic agent. DM4 is represented by the followingstructural formula (IV):

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN^(2′)-deacetyl-N-^(2′)(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the following structural formula (V):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of 5,208,020 and 7,276,697 isincorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In some embodiments, the C-3 position servesas the position to chemically link the linking moiety, and in someparticular embodiments, the C-3 position of maytansinol serves as theposition to chemically link the linking moiety.

Structural representations of some conjugates are shown below:

Several descriptions for producing such antibody-maytansinoid conjugatesare provided in U.S. Pat. Nos. 6,333,410, 6,441,163, 6,716,821, and7,368,565, each of which is incorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer can be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate can then be purified by gel filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. The average number of maytansinoidmolecules/antibody can be, for example, 1-10 or 2-5.

Anthracycline compounds, as well as derivatives, intermediates andmodified versions thereof, can also be used to prepare anti-EGFRimmunoconjugates. For example, doxorubicin, doxorubicin derivatives,doxorubicin intermediates, and modified doxorubicins can be used inanti-EGFR conjugates. Exemplary compounds are described in WO2010/009124, which is herein incorporated by reference in its entirety.Such compounds include, for example, compounds of the following formula:

wherein R₁ is a hydrogen atom, hydroxy or methoxy group and R₂ is aC₁-C_(s) alkoxy group, or a pharmaceutically acceptable salt thereof.

Conjugates of antibodies with maytansinoid or other drugs can beevaluated for their ability to suppress proliferation of variousunwanted cell lines in vitro. For example, cell lines such NCI-H226,NCI-H292, and NCI-H322M, can easily be used for the assessment ofcytotoxicity of these compounds. Cells to be evaluated can be exposed tothe compounds for 4 to 5 days and the surviving fractions of cellsmeasured in direct assays by known methods. IC₅₀ values can then becalculated from the results of the assays.

The immunoconjugates can, according to some embodiments describedherein, be internalized into cells. The immunoconjugate, therefore, canexert a therapeutic effect when it is taken up by, or internalized, by aEGFR-expressing cell. In some particular embodiments, theimmunoconjugate comprises an antibody, antibody fragment, orpolypeptide, linked to a cytotoxic agent by a cleavable linker, and thecytotoxic agent is cleaved from the antibody, antibody fragment, orpolypeptide, wherein it is internalized by a EGFR-expressing cell.

In some embodiments, the immunoconjugates are capable of reducing tumorvolume. For example, in some embodiments, treatment with animmunoconjugate results in a % T/C value that is less than about 50%,less than about 45%, less than about 40%, less than about 35%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, or less than about 5%.

In another aspect of the invention siRNA molecules can be linked to theantibodies of the present invention instead of a drug. siRNAs can belinked to the antibodies of the present invention by methods commonlyused for the modification of oligonucleotides (see, for example, USPatent Publications 20050107325 and 20070213292). Thus the siRNA in its3′ or 5′-phosphoroamidite form can be reacted with one end of thecrosslinker bearing a hydroxyl functionality to give an ester bondbetween the siRNA and the crosslinker. Similarly reaction of the siRNAphosphoramidite with a crosslinker bearing a terminal amino groupresults in linkage of the crosslinker to the siRNA through an amine.Alternatively, the siRNA can be derivatized by standard chemical methodsto introduce a thiol group. This thiol-containing siRNA can be reactedwith an antibody that has been modified to introduce an active disulfideor maleimide moiety, to produce a cleavable or non-cleavable conjugate.Between 1-20 siRNA molecules can be linked to an antibody by thismethod.

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds EGFR or a fragment of such a polypeptide. For example, theinvention provides a polynucleotide comprising a nucleic acid sequencethat encodes an antibody to a human EGFR or encodes a fragment of suchan antibody. The polynucleotides of the invention can be in the form ofRNA or in the form of DNA. DNA includes cDNA, genomic DNA, and syntheticDNA; and can be double-stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:1-38 and 69-76.

The invention further provides a polynucleotide comprising a sequenceselected from those shown in Tables 7-9 below.

TABLE 7 Variable heavy chain polynucleotide sequences AntibodyVH Polynucleotide Sequence muEGFR-caggttcagctccagcagtctggggctgagctggcaagacctggggcttcagtgaagttgtcctgcaagg7 V_(H)cttctggctacacctttactagctactggatgcagtgggtaaaacagaggcctggacagggtctggaatgtattgggactatttatcctggagatggtgatactacgtacactcagaagttcaagggcaaggccacattgactgcagataaatcctccagcacagcctacatgcaactcagcagcttggcatctgaggactctgcggtctattactgtgcaagatatgatgcccccggctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 39) muEGFR-caggttcagctccagcagtctgggactgagctggcaagacctggggcttcagtgaagttgtcctgcaagg12 V_(H)cttctggctacacctttactagctactggatgcagtgggtaaaacagaggcctggacagggtctggaatgtattgggactatctatcctggagatggtgatactaggtacattcagaagttcaagggcaaggccacattgactgcagataaatcctccagcacagcctacatgcaactcagcagcttggcatctgaggactctgcggtctattactgtgcaagatatgatgcccccggctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 40) huEGFR-aagcttgccaccatgggctggtcatgtatcattctgttcctggtggccaccgcaaccggtgtccattccca7 V_(H)ggtgcagctcgtgcagagcggggctgaagtggccaagccaggtgcttctgtcaaattgtcttgtaaggccagtgggtacaccttcacaagctactggatgcagtgggttaagcaacgcccaggccagggactggagtgcatcggcaccatttatccaggggatggagataccacttatacacaaaagtttcaaggcaaagccaccctgaccgccgacaaatccagcagcacagcatacatgcagctttctagcctcaggtctgaagactccgccgtgtactattgtgcccgctacgacgcccccggctatgcaatggattactggggccagggtactctggtcacagtgtcctccgcctctacaaagggccc (SEQ ID NO: 41) huEGFR-aagcttgccaccatggggtggtcctgtataatactgtttctggtggccactgccacaggagtccacagccaag7tgcagctggtgcagagtggcgctgaggtcaagaagcctggggcatccgtcaaggtttcttgtaaggcatctggV_(H)_CDRatataccttcacttcctattggatgcagtgggtgagacaggcaccaggacagggactggagtggatgggcactgraftedatttatccaggtgacggtgacactacttatactcagaaattcaaggggcgagtgaccatgactcgtgatactagcactagtaccgtgtatatggagcttagttctctccggtccgaggacacagcagtctactactgtgctagatatgacgcacccggatatgccatggactattgggggcagggcaccctggtcaccgtgagttccgccagcactaagggccc(SEQ ID NO: 42) huEGFR-aagcttgccaccatgggctggtcctgtattatcctctttttggtggccactgctaccggcgtacacagtcaggtgc12 V_(H)agctggtgcagtccggggctgaagtggcaaagcccggggcctccgtaaagctctcttgcaaggcatccggctacacttttacttcctactggatgcagtgggtcaaacagcgcccaggacaggggttggaatgtataggtacaatctatcccggcgatggtgacacacgatatatccagaagttccagggcaaggctaccctgactgccgacaaatcttctagcaccgcttatatgcagctgtcatctcttcgaagtgaagactctgcagtgtattactgcgcccgatatgacgcacccggttacgccatggattactggggtcaggggaccttggtaaccgtatcaagcgccagtaccaagggccc(SEQ ID NO: 43) muEGFR-caggttcagctccagcagtctggggctgagctggcaagacctggggcttcagtgaag 6 V_(H)ttgtcctgcaaggcttctggctacacctttactagctactggatgcagtgggtaaaacagaggcctggacagggtctggaatgtattggggctctttatcctggagatggtgatgctaggtacactcagaaattcaagggcaaggccacattgactgcagatagatcctccagcacagcctacatgcaactcagcagcttggcatctgaggactctgcggtctattactgtgcaagatatgatgcccccggctatgctatggactactggggtcaaggaacctcagtcaccgtcgcctca (SEQ ID NO: 77) huEGFR-6aagcttgccaccatggggtggagttgtatcatcctcttccttgtcgctaccgccactggagtgcattcccaggtgV_(H) v1.0cagttggtgcaatctggcgccgaggtggccaagcccggtgcctccgtaaaattgagttgtaaagcctctggctatacatttacatcttattggatgcagtgggtcaagcagcgccctggtcaaggcctggagtgcatcggagctctgtatcctggcgacggggacgcccgttacactcagaaatttcagggcaaagctaccctcaccgcagatacatccagcagcactgcttatatgcaacttagtagcctccgcagcgaggatagtgccgtgtactactgtgccagatatgacgccccaggttatgctatggactactggggtcaaggaaccctggtgacagtgtcaagcgctagcacaaagggccc(SEQ ID NO: 78) huEGFR-6aagcttgccaccatggggtggagttgtatcatcctcttccttgtcgctaccgccactggagtgcattcccaggtgcaV_(H) v1.11gttggtgcaatctggcgccgaggtggccaagcccggtgcctccgtaaaattgagttgtaaagcctctggctatacatttacatcttattggatgcagtgggtcaagcagcgccctggtcaaggcctggagtggatcggagctctgtatcctggcgacggggacgcccgttacactcagaaatttcagggcaaagctaccctcaccgcagatacatccagcagcactgcttatatgcaacttagtagcctccgcagcgaggatagtgccgtgtactactgtgccagatatgacgccccaggttatgctatggactactggggtcaaggaaccctggtgacagtgtcaagcgctagcacaaagggccc(SEQ ID NO: 79) huEGFR-7aagcttgccaccatgggctggtcatgtatcattctgttcctggtggccaccgcaaccggtgtccattcccaggtgcagV_(H) v1.11ctcgtgcagagcggggctgaagtggccaagccaggtgcttctgtcaaattgtcttgtaaggccagtgggtacaccttcacaagctactggatgcagtgggttaagcaacgcccaggccagggactggagtggatcggcaccatttatccaggggatggagataccacttatacacaaaagtttcaaggcaaagccaccctgaccgccgacaaatccagcagcacagcatacatgcagctttctagcctcaggtctgaagactccgccgtgtactattgtgcccgctacgacgcccccggctatgcaatggattactggggccagggtactctggtcacagtgtcctccgcctctacaaagggccc(SEQ ID NO: 80)

TABLE 8 Variable light chain polynucleotide sequences AntibodyVL Polynucleotide Sequence muEGFR-gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgca7 V_(L)aggcaagccaagacattaacaactatttggcttggtaccaacacaagcctggaaaaggtcctaggctgctcatacattacacatctacattacatccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgaagatattgcaacttattattgtctacagtatgataatcttctgtacacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 44) muEGFR-gatgtccagataacccagtctccatcttatcttgctgcatctcctggagaaaccattactattaattgca12 V_(L)gggcaagtaagagcattagcaaatatttagcctggtatcaagagaaacctgggaaaactaataagcttcttatctactctggatccactttgcaatctggaattccatcaaggttcagtggcagtggatctggtacagatttcactctcaccatcagtagcctggagcctgaagattttgcaatgtattactgtcaacagcataatgaatacccgtggacgttcggtggaggcaccaagctggaaatcaaacgg (SEQ ID NO: 45) huEGFR-gaattcgccaccatgggctggagctgcatcatcttgttcttggtcgccactgccacaggagtgcatagcgata7 V_(L) v1.0ttcagatgacccagtctcccagctctctgagcgctagcgtgggcgatcgggtgactattacttgccgtgcatcccaggatatcaacaactacttggcctggtaccagcacaagcccggcaaaggcccaaagctgctgatccactataccagtacactgcaccctggtatcccttctagattcagcggctccggtagtggtcgggattactcattctctatctcttccctggagcccgaggatatagctacatattattgtctccagtacgataatctcttgtacacatttggacaggggacaaagctggagatcaagcgtacg (SEQ ID NO: 46) huEGFR-gaattcgccaccatgggatggtcctgcattatccttttcctggtcgccaccgccacaggcgtccactctgacata7 V_(L)caaatgacccagtccccttcttcactgagcgcctccgttggggatagagttacaatcacttgtaaagctagccagv1.01gacatcaacaactatctggcttggtatcagcataaacctgggaagggacccaagctcttgattcattacacctctaccttgcacccaggcataccaagccgctttagcggtagtggcagtggccgcgattactcattctccatcagttccttggaaccagaagatatagccacctattattgtctccagtatgataatttgctctacacttttggccagggcaccaaacttgagatcaagcgtacg (SEQ ID NO: 47) huEGFR-gaattcgccaccatgggatggagttgcattattttgtttctggtagctaccgctacaggcgttcatagcgacattca7gatgacacagagcccctcctctttgtccgcctccgtgggcgatagagtcacaatcacctgccgcgcaagccagV_(L)_CDRgatatcaacaactaccttgcatggtaccagcagaagcctggaaaagccccaaagctgctcatatactacacctcgraftedcacccttcacccaggagttccatccaggttctctgggtctggaagtggaacagattttaccttcacaatcagctcattgcaacccgaggacatagctacatattactgcctgcagtatgacaatctgctgtacacatttggacagggaaccaaagttgaaatcaagcgtacg (SEQ ID NO: 48) huEGFR-gaattcgccaccatgggctggagttgcatcatcctgttcttggttgctaccgcaaccggagtacactccgacgt12 V_(L)gcagatcacccaatctccatcatccctcgccgccagtgtgggagaacgaattactatcaactgccgagcaagcv1.0cagagtatcagccgttatctggcatggtaccaggagaaacccggtaagactaacaaactgttgatttactcaggcagtacactgcaatctggtatccctagccgctttagcggctccggcagtggcaccgatttcaccctgacaatttcctccctggagccagaggatttcgcaatgtattattgtcagcaacacaacgagtacccatggacatttggccagggcacaaagctggagattaagcgtacg (SEQ ID NO: 49) huEGFR-gaattcgccaccatgggatggtcctgcattatcctgttcctcgtggcaacagctacaggggtgcatagcgatgtg12 V_(L)cagatcacccagtccccaagctcccttgcagcttccgttggtgagcgcattaccatcaactgtcgagctagtaagv1.01tctatttccaagtacctggcttggtatcaagagaagccaggaaagacaaacaagctgctcatttacagtggctctacccttcagtccggtattccctctagatttagtggcagtggtagtggaaccgattttacccttacaattagctctctggaaccagaagacttcgcaatgtactactgccagcaacacaatgagtacccatggacttttggccagggaacaaagctggaaattaaacgtacg (SEQ ID NO: 50) muEGFR-gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatc 6 V_(L)acttgcaaggcaagccaagacattaacaactatatagcttggtaccaacacaagcctggaaaaggtcctaggctgctcattcattacacatctacattacatccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgaagatattgcaacttattattgtctacagtatgataatcttctgtacacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 81)

TABLE 9 Full-length heavy and light chain polynucleotide sequencesAntibody Polynucleotide Sequence huEGFR-aagcttgccaccatgggctggtcatgtatcattctgttcctggtggccaccgcaaccggtgtccattcccaggtg7 HCcagctcgtgcagagcggggctgaagtggccaagccaggtgcttctgtcaaattgtcttgtaaggccagtgggtacaccttcacaagctactggatgcagtgggttaagcaacgcccaggccagggactggagtgcatcggcaccatttatccaggggatggagataccacttatacacaaaagtttcaaggcaaagccaccctgaccgccgacaaatccagcagcacagcatacatgcagctttctagcctcaggtctgaagactccgccgtgtactattgtgcccgctacgacgcccccggctatgcaatggattactggggccagggtactctggtcacagtgtcctccgcctctacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 51) huEGFR-gaattcgccaccatgggctggagctgcatcatcttgttcttggtcgccactgccacaggagtgcatagcgatattc7 LC v1.0agatgacccagtctcccagctctctgagcgctagcgtgggcgatcgggtgactattacttgccgtgcatcccaggatatcaacaactacttggcctggtaccagcacaagcccggcaaaggcccaaagctgctgatccactataccagtacactgcaccctggtatcccttctagattcagcggctccggtagtggtcgggattactcattctctatctcttccctggagcccgaggatatagctacatattattgtctccagtacgataatctcttgtacacatttggacaggggacaaagctggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 52) huEGFR-gaattcgccaccatgggatggtcctgcattatccttttcctggtcgccaccgccacaggcgtccactctgacatac7 LCaaatgacccagtccccttcttcactgagcgcctccgttggggatagagttacaatcacttgtaaagctagccaggav1.01catcaacaactatctggcttggtatcagcataaacctgggaagggacccaagctcttgattcattacacctctaccttgcacccaggcataccaagccgctttagcggtagtggcagtggccgcgattactcattctccatcagttccttggaaccagaagatatagccacctattattgtctccagtatgataatttgctctacacttttggccagggcaccaaacttgagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 53) huEGFR-aagcttgccaccatggggtggtcctgtataatactgtttctggtggccactgccacaggagtccacagccaag7tgcagctggtgcagagtggcgctgaggtcaagaagcctggggcatccgtcaaggtttcttgtaaggcatctgHC_CDRgatataccttcacttcctattggatgcagtgggtgagacaggcaccaggacagggactggagtggatgggcgraftedactatttatccaggtgacggtgacactacttatactcagaaattcaaggggcgagtgaccatgactcgtgatactagcactagtaccgtgtatatggagcttagttctctccggtccgaggacacagcagtctactactgtgctagatatgacgcacccggatatgccatggactattgggggcagggcaccctggtcaccgtgagttccgccagcactaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 54) huEGFR-gaattcgccaccatgggatggagttgcattattttgtttctggtagctaccgctacaggcgttcatagcgacatt7cagatgacacagagcccctcctctttgtccgcctccgtgggcgatagagtcacaatcacctgccgcgcaagLC_CDRccaggatatcaacaactaccttgcatggtaccagcagaagcctggaaaagccccaaagctgctcatatactgraftedacacctccacccttcacccaggagttccatccaggttctctgggtctggaagtggaacagattttaccttcacaatcagctcattgcaacccgaggacatagctacatattactgcctgcagtatgacaatctgctgtacacatttggacagggaaccaaagttgaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 55) huEGFR-aagcttgccaccatgggctggtcctgtattatcctctttttggtggccactgctaccggcgtacacagtcagg12 HCtgcagctggtgcagtccggggctgaagtggcaaagcccggggcctccgtaaagctctcttgcaaggcatccggctacacttttacttcctactggatgcagtgggtcaaacagcgcccaggacaggggttggaatgtataggtacaatctatcccggcgatggtgacacacgatatatccagaagttccagggcaaggctaccctgactgccgacaaatcttctagcaccgcttatatgcagctgtcatctcttcgaagtgaagactctgcagtgtattactgcgcccgatatgacgcacccggttacgccatggattactggggtcaggggaccttggtaaccgtatcaagcgccagtaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 56) huEGFR-gaattcgccaccatgggctggagttgcatcatcctgttcttggttgctaccgcaaccggagtacactccgacg12 LCtgcagatcacccaatctccatcatccctcgccgccagtgtgggagaacgaattactatcaactgccgagcaav1.0gccagagtatcagccgttatctggcatggtaccaggagaaacccggtaagactaacaaactgttgatttactcaggcagtacactgcaatctggtatccctagccgctttagcggctccggcagtggcaccgatttcaccctgacaatttcctccctggagccagaggatttcgcaatgtattattgtcagcaacacaacgagtacccatggacatttggccagggcacaaagctggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 57) huEGFR-gaattcgccaccatgggatggtcctgcattatcctgttcctcgtggcaacagctacaggggtgcatagcgatgt12 LCgcagatcacccagtccccaagctcccttgcagcttccgttggtgagcgcattaccatcaactgtcgagctagtav1.01agtctatttccaagtacctggcttggtatcaagagaagccaggaaagacaaacaagctgctcatttacagtggctctacccttcagtccggtattccctctagatttagtggcagtggtagtggaaccgattttacccttacaattagctctctggaaccagaagacttcgcaatgtactactgccagcaacacaatgagtacccatggacttttggccagggaacaaagctggaaattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 58)huEGFR-aagcttgccaccatggggtggagttgtatcatcctcttccttgtcgctaccgccactggagtgcattcccaggtg6 HC v1.0cagttggtgcaatctggcgccgaggtggccaagcccggtgcctccgtaaaattgagttgtaaagcctctggctatacatttacatcttattggatgcagtgggtcaagcagcgccctggtcaaggcctggagtgcatcggagactgtatcctggcgacggggacgcccgttacactcagaaatttcagggcaaagctaccctcaccgcagatacatccagcagcactgcttatatgcaacttagtagcctccgcagcgaggatagtgccgtgtactactgtgccagatatgacgccccaggttatgctatggactactggggtcaaggaaccctggtgacagtgtcaagcgctagcacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagaggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 82) huEGFR-aagcttgccaccatggggtggagttgtatcatcctcttccttgtcgctaccgccactggagtgcattcccaggtgca6 HCgttggtgcaatctggcgccgaggtggccaagcccggtgcctccgtaaaattgagttgtaaagcctctggctatacatv1.11ttacatcttattggatgcagtgggtcaagcagcgccctggtcaaggcctggagtggatcggagctctgtatcctggcgacggggacgcccgttacactcagaaatttcagggcaaagctaccctcaccgcagatacatccagcagcactgcttatatgcaacttagtagcctccgcagcgaggatagtgccgtgtactactgtgccagatatgacgccccaggttatgctatggactactggggtcaaggaaccctggtgacagtgtcaagcgctagcacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 83) huEGFR-aagcttgccaccatgggctggtcatgtatcattctgttcctggtggccaccgcaaccggtgtccattcccaggtgcagct7 HCcgtgcagagcggggctgaagtggccaagccaggtgcttctgtcaaattgtcttgtaaggccagtgggtacaccttcacav1.11agctactggatgcagtgggttaagcaacgcccaggccagggactggagtggatcggcaccatttatccaggggatggagataccacttatacacaaaagtttcaaggcaaagccaccctgaccgccgacaaatccagcagcacagcatacatgcagctttctagcctcaggtctgaagactccgccgtgtactattgtgcccgctacgacgcccccggctatgcaatggattactggggccagggtactctggtcacagtgtcctccgcctctacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccaccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 84)

Also provided is a polynucleotide having at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NOs:39-58, 77-84. Thus, in certainembodiments, the polypeptide comprises (a) a polypeptide having at leastabout 95% sequence identity to SEQ ID NOs:39-43, 51, 54, 56, 77-80 or82-84 and/or (b) a polypeptide having at least about 95% sequenceidentity to SEQ ID NOs:44-50, 52, 53, 55, 57, 58, 81. In certainembodiments, the polypeptide comprises (a) a polypeptide having theamino acid sequence of SEQ ID NOs: 39-43, 51, 54, 56, 77-80 or 82-84;and/or (b) a polypeptide having the amino acid sequence of SEQ ID NOs:44-50, 52, 53, 55, 57, 58, 81.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

IV. Methods of Use and Pharmaceutical Compositions

The EGFR-binding agents (including antibodies, immunoconjugates, andpolypeptides) of the invention are useful in a variety of applicationsincluding, but not limited to, therapeutic treatment methods, such asthe treatment of cancer. In certain embodiments, the agents are usefulfor inhibiting tumor growth, inducing differentiation, reducing tumorvolume, and/or reducing the tumorigenicity of a tumor. The methods ofuse can be in vitro, ex vivo, or in vivo methods. In certainembodiments, the EGFR-binding agent or antibody or immunoconjugate, orpolypeptide is not antagonistic of the human EGFR to which it binds.

In one aspect, anti-EGFR antibodies and immunoconjugates of theinvention are useful for detecting the presence of EGFR in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue.

In one aspect, the invention provides a method of detecting the presenceof EGFR in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-EGFR antibodyunder conditions permissive for binding of the anti-EGFR antibody toEGFR, and detecting whether a complex is formed between the anti-EGFRantibody and EGFR.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of EGFR. In certain embodiments,the method comprises contacting a test cell with an anti-EGFR antibody;determining the level of expression (either quantitatively orqualitatively) of EGFR by the test cell by detecting binding of theanti-EGFR antibody to EGFR; and comparing the level of expression ofEGFR by the test cell with the level of expression of EGFR by a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses EGFR at levels comparable to such a normal cell),wherein a higher level of expression of EGFR by the test cell ascompared to the control cell indicates the presence of a disorderassociated with increased expression of EGFR. In certain embodiments,the test cell is obtained from an individual suspected of having adisorder associated with increased expression of EGFR. In certainembodiments, the disorder is a cell proliferative disorder, such as acancer or a tumor.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-EGFRantibody to EGFR expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing EGFR on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-EGFR antibody under conditions permissive for binding of theanti-EGFR antibody to EGFR, and detecting whether a complex is formedbetween the anti-EGFR antibody and EGFR on the cell surface. Anexemplary assay for detecting binding of an anti-EGFR antibody to EGFRexpressed on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-EGFRantibodies to EGFR. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-EGFR antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction.

In certain embodiments, anti-EGFR antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-EGFRantibody from any EGFR that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-EGFRantibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-EGFR antibody afterformation of a complex between the anti-EGFR antibody and EGFR, e.g., byimmunoprecipitation.

Any of the above embodiments of diagnosis or detection can be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-EGFR antibody.

In certain embodiments, the disease treated with the EGFR-binding agentor antagonist (e.g., an anti-EGFR antibody) is a cancer. In certainembodiments, the cancer is characterized by EGFR expressing cells towhich the EGFR-binding agent (e.g., antibody) binds.

In a further aspect, the invention is directed to an improved method fortreating cell proliferation disorders wherein EGFR is expressed,particularly wherein EGFR is abnormally expressed (e.g. overexpressed),including cancers of the bladder, brain, head and neck, pancreas, lung,breast, ovary, colon, prostate, skin, and kidney, comprisingadministering a therapeutically effective amount of an anti-EGFR bindingagent of the present invention to a human subject in need thereof. Inanother embodiment the antibody is humanized. Examples of cellproliferation disorders that can be treated by an anti-EGFR bindingagent of the present invention include, but are not limited to neoplasmslocated in the: abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system.

Similarly, other cell proliferation disorders can also be treated by theanti-EGFR binding agents of the present invention. Examples of such cellproliferation disorders include, but are not limited to: adrenal cortexhyperplasia (Cushing's disease), congenital adrenal hyperplasia,endometrial hyperplasia, benign prostatic hyperplasia, breasthyperplasia, intimal hyperplasia, focal epithelial hyperplasia (Heck'sdisease), sebaceous hyperplasia, compensatory liver hyperplasia, and anyother cell proliferation disease, besides neoplasia, located in an organsystem listed above.

The present invention further provides methods for inhibiting tumorgrowth using the antibodies or other agents described herein. In certainembodiments, the method of inhibiting the tumor growth comprisescontacting the cell with an EGFR-binding agent (e.g., antibody) invitro. For example, an immortalized cell line or a cancer cell line thatexpresses EGFR is cultured in medium to which is added the antibody orother agent to inhibit tumor growth. In some embodiments, tumor cellsare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and cultured in medium towhich is added an EGFR-binding agent to inhibit tumor growth.

In some embodiments, the method of inhibiting tumor growth comprisescontacting the tumor or tumor cells with the EGFR-binding agent (e.g.,antibody) in vivo. In certain embodiments, contacting a tumor or tumorcell with a EGFR-binding agent is undertaken in an animal model. Forexample, EGFR-binding agents can be administered to xenograftsexpressing one or more EGFRs that have been grown in immunocompromisedmice (e.g. NOD/SCID mice) to inhibit tumor growth. In some embodiments,cancer stem cells are isolated from a patient sample such as, forexample, a tissue biopsy, pleural effusion, or blood sample and injectedinto immunocompromised mice that are then administered a EGFR-bindingagent to inhibit tumor cell growth. In some embodiments, theEGFR-binding agent is administered at the same time or shortly afterintroduction of tumorigenic cells into the animal to prevent tumorgrowth. In some embodiments, the EGFR-binding agent is administered as atherapeutic after the tumorigenic cells have grown to a specified size.

In certain embodiments, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of aEGFR-binding agent. In certain embodiments, the subject is a human. Incertain embodiments, the subject has a tumor or has had a tumor removed.

In certain embodiments, the tumor expresses the EGFR to which theEGFR-binding agent or antibody binds.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering atherapeutically effective amount of a EGFR-binding agent to the subject.In certain embodiments, the tumor comprises cancer stem cells. Incertain embodiments, the frequency of cancer stem cells in the tumor isreduced by administration of the agent.

The invention further provides methods of differentiating tumorigeniccells into non-tumorigenic cells comprising contacting the tumorigeniccells with a EGFR-binding agent (for example, by administering theEGFR-binding agent to a subject that has a tumor comprising thetumorigenic cells or that has had such a tumor removed.

The use of the EGFR-binding agents, polypeptides, or antibodiesdescribed herein to induce the differentiation of cells, including, butnot limited to tumor cells, is also provided. For example, methods ofinducing cells to differentiate comprising contacting the cells with aneffective amount of a EGFR-binding agent (e.g., an anti-EGFR antibody)described herein are envisioned. Methods of inducing cells in a tumor ina subject to differentiate comprising administering a therapeuticallyeffective amount of a EGFR-binding agent, polypeptide, or antibody tothe subject are also provided. In certain embodiments, the tumor is apancreatic tumor. In certain other embodiments, the tumor is a colontumor. In some embodiments, the treatment methods comprise administeringa therapeutically effective amount of the EGFR-binding agent,polypeptide, or antibody to the subject.

The present invention further provides pharmaceutical compositionscomprising one or more of the EGFR-binding agents described herein. Incertain embodiments, the pharmaceutical compositions further comprise apharmaceutically acceptable vehicle. These pharmaceutical compositionsfind use in inhibiting tumor growth and treating cancer in humanpatients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g. carrier, excipient)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight polypeptides (e.g. less than about 10 amino acid residues);proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; carbohydratessuch as monosaccharides, disaccharides, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); and non-ionic surfactants such asTWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

An antibody or immunoconjugate of the invention can be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen can have complementary activities to the ADC of the combinationsuch that they do not adversely affect each other. Pharmaceuticalcompositions comprising the EGFR-binding agent and the secondanti-cancer agent are also provided.

For the treatment of the disease, the appropriate dosage of an antibodyor agent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antibody or agent is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g. reduction in tumor size). Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantibody or agent. The administering physician can easily determineoptimum dosages, dosing methodologies and repetition rates. In certainembodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, andcan be given once or more daily, weekly, monthly or yearly. In certainembodiments, the antibody or other EGFR-binding agent is given onceevery two weeks or once every three weeks. In certain embodiments, thedosage of the antibody or other EGFR-binding agent is from about 0.1 mgto about 20 mg per kg of body weight. The treating physician canestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues.

The combination therapy can provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

VI. Kits Comprising EGFR Binding Agents

The present invention provides kits that comprise the antibodies,immunoconjugates or other agents described herein and that can be usedto perform the methods described herein. In certain embodiments, a kitcomprises at least one purified antibody against EGFR in one or morecontainers. In some embodiments, the kits contain all of the componentsnecessary and/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results. One skilled in the art willreadily recognize that the disclosed antibodies, immunoconjugates orother agents of the present invention can be readily incorporated intoone of the established kit formats which are well known in the art.

Further provided are kits comprising a EGFR-binding agent (e.g., aEGFR-binding antibody), as well as a second anti-cancer agent. Incertain embodiments, the second anti-cancer agent is a chemotherapeuticagent (e.g., rituximab).

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1

Production of Murine EGFR Antibodies

To produce murine anti-EGFR antibodies, head and neck squamous carcinomacell lines such as CA922 (Japanese Collection of Research Bioresources(JCRB), Shinjuku, Japan) and HSC4 (JCRB, Shinjuku, Japan) were injectedsubcutaneously into Balb/c female mice (Charles River Laboratory,Wilmington, Mass.) at the dose of 5×10⁶ cells per mouse every 2 weeksfor 5 times. Three days before being sacrificed for hybridomageneration, the immunized mice received intraperitoneal injection ofanother dose of antigen. The spleen from the mouse was collectedaccording to standard animal protocols and was ground between twosterile, frosted microscopic slides to obtain a single cell suspensionin RPMI-1640 medium. After the red blood cells were lysed with ACKlysing buffer, the spleen cells were then mixed with murine myelomaP3X63Ag8.653 cells (P3 cells) (J. F. Kearney et al. 1979, J Immunol,123:1548-1550) at ratio of 1 P3 cells:3 spleen cells. The mixture ofspleen cells and P3 cells was washed and treated with pronase in fusionmedia (0.3M mannitol/D-sorbitol, 0.1 mM CaCl2, 0.5 mM MgCl2 and 1 mg/mlBSA) at room temperature for 3 min. The reaction was stopped by additionof FBS (Fetal Bovine Serum, Invitrogen); cells were then washed,resuspended in 2 ml cold fusion media and fused with BTX ECM 2001electrofusion machine (Harvard Apparatus). The fused cells were addedgently to RPMI-1640 selection medium containinghypoxanthine-aminopterin-thymidine (HAT) (Sigma Aldrich), incubated for20 min at 37° C., and then seeded into flat bottom 96-well plates at 200μl/well. The plates were then incubated in 5% CO₂ incubator at 37° C.until hybridoma clones were ready for antibody screening. Othertechniques of immunization and hybridoma production can also be used,including those described in J. Langone and H. Vunakis (Eds., Methods inEnzymology, Vol. 121, “Immunochemical Techniques, Part I”; AcademicPress, Florida) and E. Harlow and D. Lane (“Antibodies: A LaboratoryManual”; 1988; Cold Spring Harbor Laboratory Press, New York, N.Y.).

Murine Hybridoma Screening and Selection

Hybridoma screening was done using flow cytometric binding assay withimmunizing cells that are either untreated or treated with EGF (R&DSystems). Because incubation with EGF downregulates EGFR level on thecell surface, EGFR specific hybridoma supernatant would only react tothe untreated cells and not to the EGF-treated cells. Cells forscreening were first cultured in serum free media for overnight andseparated into two parts. One part of the cells was left untreated andthe other part was treated with EGF for 3 hours at 37° C. The EGFtreated cells were labeled with CellTrace™ far red DDAO-SE (Invitrogen),mixed with untreated cells at 1:1 ratio and incubated with the hybridomasupernatant for 2 hours on ice. Cells were then washed, incubated withFITC-labeled anti-mouse IgG (Jackson Immunoresearch), washed, fixed withformalin and analyzed using FACScalibur (BD Bioscience). The EGFRspecific hybridomas were expanded and the supernatants were rescreenedby ELISA using soluble recombinant human EGFR extracellular domain(RELIATech) as antigen. The positive hybridomas were rescreened usingflow cytometric binding assay with human EGFR-expressing A431 epidermalcarcinoma cell line (ATCC) and monkey EGFR-expressing Vero cell line (anAfrican green monkey kidney epithelial cell line) (ATCC). In brief, thehybridoma supernatant was incubated with A431 cells and DDAO-labeledVero cells on ice for 1 hour. The cells were washed twice and incubatedwith PE-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch)for 1 hour on ice. The cells were then washed with FACS buffer, fixed informalin and analyzed using a FACSCalibur flow cytometer (BDBiosciences).

The positive hybridoma clones that reacted to both human and monkeyantigens were tested for the capacity to inhibit basal proliferation ofEGFR-overexpressing HCC827 cells (ATCC). In brief, exponentially growingHCC827 cells were plated at 2000 cells/well in 96 well plates in 100 μlcomplete media containing 10% FBS. 100 μl of hybridoma supernatant wasadded to the cells and the mixture was incubated at 37° C. in ahumidified 5% CO₂ incubator for 5 days. Level of cell proliferation wasdetermined using colorimetric WST-8 assay (Dojindo MolecularTechnologies, Rockville, Md.). WST-8 is reduced by dehydrogenases in theliving cells to an orange formazan product that is soluble in tissueculture medium, and the amount of formazan produced is directlyproportional to the number of living cells. 10% of the final volume ofWST-8 was added to each well and plates were incubated at 37° C. for anadditional 2-4-h. Plates were then analyzed by measuring the absorbanceat 450 nm (A450) in the Spectra Max M2 plate reader (Molecular Devices,Sunnyvale, Calif.). Background A450 absorbance of wells with media andWST-8 only was subtracted from all values. The surviving fraction wascalculated by dividing each treated sample value by the average value ofwells with untreated cell (surviving fraction=(A450 treated sample−A450background)/(A450 untreated sample−A450 background)). The results werenormalized so that surviving fraction 0 indicated the value of wellswithout cells and 1 indicated the level of cell proliferation in theserum containing media without any EGFR antibody. Hybridoma clones thatinhibited at least 50% HCC827 cell growth were subcloned by limitingdilution. One subclone from each hybridoma, which showed the samereactivity against EGFR as the parental cells by flow cytometry, waschosen for subsequent analysis. Stable subclones were cultured and theantibody was isotyped using commercial isotyping reagents (Roche).

Antibody Purification

Antibodies were purified from hybridoma subclone supernatants usingstandard methods, such as, for example Protein A or G chromatography(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,supernatant was prepared for chromatography by the addition of 1/10volume of 1M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant wasfiltered through a 0.22 μm filter membrane and loaded onto columnequilibrated with binding buffer (PBS, pH 7.3). The column was washedwith binding buffer until a stable baseline was obtained with noabsorbance at 280 nm. Antibody was eluted with 0.1M acetic acid buffercontaining 0.15M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.Fractions of approximately 0.25 mL were collected and neutralized by theaddition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) wasdialyzed overnight twice against 1×PBS and sterilized by filteringthrough a 0.2 μm filter membrane. Purified antibody was quantified byabsorbance at A280.

Protein A purified fractions were further polished using ion exchangechromatography (IEX) with quaternary ammonium (Q) chromatography formurine antibodies. Briefly, samples from protein A purification werebuffer exchanged into binding buffer (10 mM Tris, 10 mM sodium chloride,pH 8.0) and filtered through 0.22 μm filer. The prepared sample was thenloaded onto a Q fast flow resin (GE Lifesciences) that was equilibratedwith binding buffer at a flow rate of 120 cm/hr. Column size was chosento have sufficient capacity to bind all the MAb in the sample. Thecolumn was then washed with binding buffer until a stable baseline wasobtained with no absorbance at 280 nm. Antibody was eluted by initiatinga gradient from 10 mM to 500 mM sodium chloride in 20 column volume(CV). Peak fractions were collected based on absorbance measurement at280 nm (A280). The percentage of monomer was assessed with sizeexclusion chromatography (SEC) on a TSK gel G3000SWXL, 7.8×300 mm with aSWXL guard column, 6.0×40 mm (Tosoh Bioscience, Montgomeryville, Pa.)using an Agilent HPLC 1100 system (Agilent, Santa Clara, Calif.).Fractions with monomer content above 95% were pooled, buffer exchangedto PBS (pH 7.4) using a TFF system, and sterilized by filtering througha 0.2 μm filter membrane. The IgG concentration of purified antibody wasdetermined by A280 using an extinction coefficient of 1.47. Alternativemethods such as ceramic hydroxyapatite (CHT) were also used to polishantibodies with good selectivity. Type II CHT resin with 40 μm particlesize (Bio-Rad Laboratories) were used with a similar protocol asdescribed for IEX chromatography. The binding buffer for CHT correspondsto 20 mM sodium phosphate, pH 7.0 and antibody was eluted with agradient of 20-160 mM sodium phosphate over 20 CV.

Example 2 Binding Affinity to Human and Monkey EGFR Antigen

To determine the binding affinity of the EGFR antibodies to the humanand monkey antigens, EGFR expressing human tumor cell lines such asMDA-MB468 (ATCC) and A431 (ATCC), and Vero monkey kidney cell line(ATCC) were used in a flow cytometric binding assay. In brief, the cellswere incubated with various concentration of EGFR antibody for 1 h at 4°C. The cells were washed and incubated with PE-conjugated secondaryantibody (Jackson Immunoresearch) for 1 h at 4° C. The cells were thenwashed, fixed in formalin and analyzed in FACSarray (BD Bioscience). Todetermine the binding affinity of these antibodies, geometric meanfluorescence intensity was plotted against the antibody concentration ina semi-log plot. A dose-response curve was generated by non-linearregression and the EC50 value of the curve, which corresponds to theapparent dissociation constant (Kd) of each antibody, was calculatedusing GraphPad Prism v4 (GraphPad software). The EGFR antibodies of theinvention as well as positive control antibodies, cetuximab andpanitumumab, showed strong specific binding to both human tumor cellsand monkey Vero cells. A table shown in FIG. 1 lists the Kd of eachantibody to the human and monkey EGFR. The EGFR antibodies of theinvention exhibited similarly strong binding affinity to both human andmonkey antigens.

Example 3 Inhibition of Ligand-Induced EGFR Activation

EGFR ligand binding induces EGFR phosphorylation followed by activationof downstream signaling pathways. To investigate the effect of anti-EGFRantibodies in ligand-induced EGFR activation, Western blot analysis wasperformed using MDA-MB468 tumor cell line and human primary adultkeratinocytes. In brief, cells were seeded at 1e6 cells/well in a 6 wellplate and cultured in normal media for overnight. Cells were washed andstarved in serum free media containing 0.1% BSA for 2 hours at 37° C. 10μg/ml antibody was added to the cells and the mixture was incubated for3 hours at 37° C. 50 ng/ml EGF (R&D Systems) was added to the mixtureand incubated for 15 minutes at 37° C. Cells were then washed withice-cold PBS and lysed in RIPA buffer containing protease andphosphatase inhibitors. The protein lysates were separated in SDS-PAGEand transferred to a nitrocellulose membrane. The membrane was blockedwith 5% BSA and incubated with anti-phosphotyrosine antibody (clone4G10, Millipore) for overnight at 4° C. The membrane was washed,incubated with HRP conjugated anti-mouse antibody (JacksonImmunoresearch) for 1 hour at room temperature, and washed again. Thesignal was detected using an ECL (enhanced chemiluminescence) system (GEHealthcare). To ensure equal amount of proteins loaded into each lane,the membrane was stripped and reprobed with anti-β-tubulin antibody(Sigma Aldrich).

As shown in FIG. 2, EGF stimulation led to strong EGFR phosphorylationin both MDA-MB468 cells and human primary keratinocytes. Treatment ofcells with cetuximab and panitumumab strongly inhibited the EGF-inducedEGFR phosphorylation while the EGFR antibodies of the invention did notcompletely inhibit EGFR activation. Anti-KTI (Kuntz Trypsin inhibitor)antibody (produced from hybridoma obtained from ATCC) was used asnegative control.

Example 4 Agonistic Activity of EGFR Antibodies

To investigate the effect of the EGFR antibodies of the invention onEGFR signaling in absence of EGFR ligands, MDA-MB468 tumor cells werestarved in serum free media as described in Example 5. The cells werethen incubated with 10 μg/ml EGFR antibodies for 3 hours at 37° C. Aspositive control, untreated cells were incubated with 50 ng/ml EGF for15 minutes at 37° C. The protein lysates were prepared and analyzedusing Western blot as described in Example 5. The representative resultis shown in FIG. 3. EGF treatment clearly induced a strong EGFRphosphorylation in MDA-MB468 cells while the EGFR antibodies of theinvention did not affect EGFR signaling in absence of the ligand.

Example 5 Ligand Binding Competition

One mechanism of EGFR signaling inhibition is blockade of ligandbinding. To examine if the EGFR antibodies inhibit the ligand binding tothe EGFR, the binding of biotinylated EGFR ligand to the A431 cells wasmeasured by flow cytometry in the presence of anti-EGFR antibodies.Biotinylation of TGFα was done using EZ-link MicroSulfo-NHS-LC-biotinylation kit (Pierce, Rockland, Ill.) according to themanufacturer's instruction. Biotinylated EGF was obtained fromInvitrogen. Prior to competition assay, the binding curve of thebiotinylated ligands was established. Varying concentrations ofanti-EGFR antibodies were pre-mixed with biotinylated ligands at EC50concentration (1.8 nM and 10 nM for EGF and TGFa, respectively), and themixture was incubated with the cells for 30 min on ice. Cells were thenwashed twice with FACS buffer and incubated with streptavidin-APCconjugate (Jackson Immunoresearch) for 15 min on ice. Cells were washedtwice with FACS buffer and analyzed in FACS Calibur (BD Bioscience)using FlowJo program (Tree Star). The geometric mean fluorescenceintensities were plotted against the antibody concentration in asemi-log plot. As shown in FIG. 4, the negative control antibody,anti-KTI antibody does not affect the ligand binding while all anti-EGFRantibodies compete the ligand binding with the following EC50 (Table10).

TABLE 10 EC50 of TGFa binding EC50 of EGF binding competition (nM)competition (nM) Ligand 1.336 2.097 Cetuximab 0.769 1.226 EGFR-6 1.1812.552 EGFR-12 1.341 2.927 EGFR13 1.321 1.957

The EGFR antibodies of the invention completely blocked the TGFα and EGFbinding with similar EC50 as cetuximab (FIG. 4 and Table 10). Thisresult cannot explain the differential effect of cetuximab and the EGFRantibodies of the invention on ligand induced EGFR signaling (Example 3)and growth of normal epithelial cell lines including human primarykeratinocytes (Example 6).

Example 6 Growth Inhibition Assay of Human Primary Keratinocytes andNormal Epithelial Cell Line

Human normal basal epithelial cells in skin, gastrointestinal tract andother organs physiologically express EGFR. The EGFR signaling in thesetissues is critical for the epithelial cell growth. Disruption of EGFRsignaling pathway by cetuximab and panitumumab as well as small tyrosinekinase inhibitors such as erlotinib and gefitinib causes significantskin toxicity. To mimic the potential toxicity in skin and otherepithelial cells, proliferation assays using human primary keratinocytes(Invitrogen) and a non-tumorigenic breast epithelial cell line, MCF10A(ATCC), were established. In this assay, cells were plated at1,500-2,000 cells per well in EGFR ligand-containing media suggested bythe manufacturers and incubated with anti-EGFR antibodies at 37° C. for5 days. In the keratinocytes assay (FIG. 5), cells were grown inpresence of 1 nM EGF with varying concentration of antibodies. While inMCF10A cell assay (FIG. 6), cells were grown in presence of 10 nM EGFwith a fixed concentration (10 μg/ml) of antibodies. Level of cellproliferation was determined using colorimetric WST-8 assay (DojindoMolecular Technologies, Rockville, Md.) as described in example 1. Thesurviving fraction was calculated by dividing each treated sample valueby the average value of wells with untreated cell (survivingfraction=(A450 treated sample−A450 background)/(A450 untreatedsample−A450 background)). The results were normalized so that 0indicated the level of cell proliferation in absence of EGF and 1indicated the level of cell proliferation in presence of EGF without anyanti-EGFR antibody treatment.

The binding of the anti-EGFR antibodies to human primary keratinocytesas well as MCF10A cells was confirmed before the proliferation assayswere performed. A representative result of the keratinocytesproliferation assay is shown in FIG. 5. As expected from the toxicityprofile, cetuximab and panitumumab strongly inhibited the keratinocytesproliferation in dose dependent manner with the maximal inhibition of40% for cetuximab and 78% for panitumumab. Surprisingly, the EGFRantibody of the invention, similar as the negative control chimeric KTIantibody, had little or no effect on the keratinocytes. This result wasconfirmed in the MCF10A proliferation assay (FIG. 6). The cetuximab andpanitumumab strongly inhibited MCF10A cell proliferation while the EGFRantibodies of the invention had little or no effect on MCF10A cellgrowth. These data suggest that the EGFR antibodies of the invention areless toxic than cetuximab and panitumumab on normal epithelial cellsthat express EGFR.

Example 7 Inhibition of Basal Proliferation of HCC827 and NCI-H292 CellLines

To determine the capacity of anti-EGFR antibodies in inhibiting thebasal proliferation of tumor cells, proliferation assays withEGFR-expressing HCC827 (ATCC) and NCI-H292 (ATCC) lung tumor cell lineswere established. Cells were plated at 2,000 cells per well in normalgrowth media containing 10% FBS and grown at 37° C. for 5 days inpresence of varying concentration of anti-EGFR antibodies. Level of cellproliferation was determined using colorimetric WST-8 assay. The ODresults were normalized so that surviving fraction 1 represents cellsgrown in normal growth media without anti-EGFR antibodies, and 0represents the value of wells without cells.

FIGS. 7A and 7B show the representative proliferation assay results withHCC827 and NCI-H292 cells, respectively. In HCC827 cell line (FIG. 7A),cetuximab strongly inhibited the tumor cell growth in dose dependentmanner. Despite being harmless to EGFR expressing normal epithelialcells, anti-EGFR antibodies of the invention showed similar or betterinhibitory activity than cetuximab. Table 11 describes the EC50 and %maximal inhibition of each antibody.

TABLE 11 Anti-proliferative activity of anti- EGFR antibodies in HCC827cells Antibody EC50 (nM) Maximal inhibition (%) Cetuximab 0.13 65 2 0.3270 5 1.3 72 6 0.3 72 7 0.03 72 12 0.7 63 13 0.03 69 15 0.7 67 17 0.06 64

In NCI-H292 cell line (FIG. 7B), cetuximab and panitumumab stronglyinhibited the basal cell proliferation in dose dependent manner. TheEGFR antibodies of the invention were also significantly active in thiscell line with the maximal proliferation inhibition was similar tocetuximab and panitumumab. Table 12 describes the EC50 and % maximalinhibition of each antibody.

TABLE 12 Anti-proliferative activity of anti- EGFR antibodies inNCI-H292 cells Antibody EC50 (nM) Maximal inhibition (%) Panitumumab0.039 76 Cetuximab 0.035 74 2 1.3 75 5 4.7 60 6 5.6 65 7 0.4 77 13 0.478 15 4.7 75 17 1.5 67

These data strongly argue that the EGFR antibodies of the invention areas active as cetuximab and panitumumab in EGFR overexpressing cellswhile they do not affect the normal epithelial cell growth.

Example 8 Anti-EGFR Antibody Binding Competition

To distinguish binding epitopes of anti-EGFR antibodies, antibodybinding competition assays were done using flow cytometry. In thisexperiment, binding of 0.3 nM biotinylated 528 (FIG. 8A), 0.2 nMbiotinylated cetuximab (FIG. 8B) and 0.2 nM biotinylated EGFR-7 antibody(FIG. 8C) to the MDA-MB468 cells was measured in presence of varyingconcentration of ‘competing’ antibodies. In brief, the biotinylatedantibody was pre-mixed with varying concentration of ‘competing’antibodies. The antibody mixture was then incubated with cells on icefor 2 h. The cells were washed and incubated with streptavidin-alexa 488conjugate on ice for 1 h. After washing, the cells were fixed andanalyzed in FACScalibur. The geometric mean fluorescence intensity wasplotted against antibody concentration in semi-log plot and normalizedso that 100% represents the maximal binding of the biotinylated antibodyin absence of other antibody and 0% represents the background stainingin absence of the biotinylated antibody. As shown in FIG. 8, all theEGFR antibodies compete with each other. Cetuximab and panitumumabcompeted the 528 antibody binding as strong as the naked 528 antibodywhile the EGFR antibodies of the invention had slightly less capacity tocompete with 528 antibody (FIG. 8A). All EGFR antibodies competed thebinding of cetuximab and EGFR-7 biotinylated antibodies in similarmanner (FIGS. 8B and 8C).

Example 9 Cloning and Sequencing of the VL and VH Regions of the EGFRAntibodies

Total cellular RNA was prepared from 5×10⁶ cells of the EGFR hybridomasusing an RNeasy kit (QIAgen) according to the manufacturer's protocol.cDNA was subsequently synthesized from total RNA using the SuperScriptII cDNA synthesis kit (Invitrogen).

The procedure for the first round degenerate PCR reaction on the cDNAderived from hybridoma cells was based on methods described in Wang etal. ((2000) J Immunol Methods. 233:167-77) and Co et al. ((1992) JImmunol. 148:1149-54). VH sequences were amplified by PCR using thefollowing degenerate primers: EcoMH1 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC(SEQ ID NO:59), EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ IDNO:60) and BamIgG1 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO:61).VL sequences were amplified by PCR using the following degenerateprimers: SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO:62) andHindKL TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQ ID NO:85).(Mixed bases are defined as follows: N=G+A+T+C, S=G+C, Y═C+T, M=A+C,R=A+G, W=A+T). The PCR reaction mixtures were then run on a 1% low meltagarose gel, the 300 to 400 bp bands were excised, purified using ZymoDNA mini columns, and sent to Agencourt Biosciences for sequencing. Therespective 5′ and 3′ PCR primers were used as sequencing primers togenerate the variable region cDNAs from both directions. The amino acidsequences of VH and VL regions were predicted from the DNA sequencingresults.

Since the degenerate primers used to clone the VL and VH cDNA sequencesalters the 5′ end sequences, additional sequencing efforts were neededto verify the complete sequences. The preliminary cDNA sequences wereused to search the NCBI IgBlast site(http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline sequencesfrom which the antibody sequences are derived. PCR primers were thendesigned to anneal to the germline linked leader sequence of the murineantibody so that this new PCR reaction would yield a complete variableregion cDNA sequence, unaltered by the PCR primers. The PCR reactions,band purifications, and sequencing were performed as described above.

Mass Determination for Sequence Confirmation

The cDNA sequence information for the variable region was combined withthe germline constant region sequence to obtain full length antibodycDNA sequences. The molecular weights of the heavy chain and light chainwere then calculated and compared with the molecular weights obtained byLC/MS analyses of the murine EGFR antibodies. The molecular weightmeasurements are consistent with the cDNA sequences for both the EGFR-7and EGFR-12 light and heavy chains.

Composite CDR Sequences for the EGFR-7 Variants

A number of murine anti-EGFR hybridomas were sequenced and found toexpress antibodies with light and heavy chain variable region sequencesnearly identical to EGFR-7, but with some CDR amino acid substitutions,particularly in heavy chain CDR2 (FIG. 9). Since these CDR variants ofmurine EGFR-7 were found to be functionally identical, they provide somestructural insight into the sequence flexibility of the EGFR-7 CDR's.Light chain CDR's 2 and 3 as well as heavy chain CDR 1 were identical ineach of the variants while a single residue substitution was found at alow frequency in light chain CDR1 and heavy chain CDR3. As opposed tothe CDR's with an apparently tight sequence conservation, the variantsof EGFR-7 frequently contained as many as 4 amino acid substitutions inheavy chain CDR2. These sequence variants of EGFR-7 suggest that the 5tightly conserved CDR's may provide the structural basis for EGFRbinding, while heavy chain CDR2 has some sequence flexibility resultingin a somatic mutation hotspot during affinity maturation. Table 4provides a composite CDR sequence listing based on EGFR-7 variantstogether with the CDR variants described above for humanization.Humanized antibodies derived from these composite CDR's would beexpected to preserve the functional attributes of EGFR-7.

TABLE 13 EGFR-7 variant composite CDR's Light Chain CDR1:[KorR]ASQDINNY[LorI]A (SEQ ID NO: 14) CDR2: YTSTLHP (SEQ ID NO: 11)CDR3: LQYDNLLYT (SEQ ID NO: 12) Heavy Chain CDR1: TSYWMQ (SEQ ID NO: 1)CDR2: [TorA][IorL]YPGDGD[TorA][T, R, orS] (SEQ ID NO: 4) Kabat CDR2:[TorA][IorL]YPGDGD[TorA][T, R, orS][YTorI]QKF[QorK]G (SEQ ID NO: 6)CDR3: YDAPGY[AorT]MDY (SEQ ID NO: 5)

Example 10 Antibody Humanization

The EGFR-7 and EGFR-12 antibodies were humanized following resurfacingmethods previously described, such as, for example in Roguska et al.,Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al.,Protein Eng. 9(10):895-904 (1996), which are incorporated in theirentirety herein by reference. Resurfacing generally involvesidentification of the variable region framework surface residues in boththe light and heavy chains and replacing them with human equivalents.The murine CDR's are preserved in the resurfaced antibody. ExemplaryCDRs of EGFR-7 and EGFR-12 antibodies are defined as indicated in Table13. To minimize concerns about the impact of conjugating lysines thatfall in CDR's, lysine 24 in murine EGFR-7 antibody light chain CDR1 wasreplaced with arginine for humanized version 1.0 (shown in italic), soboth versions of the LC CDR1 are given. Similarly, lysine 27 and lysine31 in murine EGFR-12 antibody light chain CDR1 were replaced withglutamine and arginine, respectively, for humanized version 1.0 (shownin italic). The replacement of lysine 27 with glutamine instead ofarginine was because glutamine is reserved in human germline sequencesat this position. In addition to the AbM heavy chain CDR2 definitionemployed for resurfacing, the table provides exemplary Kabat definedheavy chain CDR2's for both the murine and human versions of EGFR-7 andEGFR-12 antibodies. The underlined sequence marks the portion of theKabat heavy chain CDR2 that was not considered a CDR for resurfacing.

Surface residue positions were defined as any position with a relativeaccessibility of 30% or greater (Pedersen J. T. et. Al, J. Mol. Biol.1994; 235: 959-973). The calculated surface residues were then alignedwith human germline surface sequences to identify the most homologoushuman surface sequence. The human germline sequence used as thereplacement surface for the light chain variable domains of EGFR-7antibody was IGKV1-12*01 while IGKV1-16*01 was used as the replacementsurface for EGFR-12 antibody VL. The human germline sequences used asthe replacement surfaces for the heavy chain variable domains of EGFR-7and EGFR-12 antibodies were IGHV1-69*02 and IGHV1-69*08, respectively.The specific framework surface residue changes for EGFR-7 and EGFR-12antibodies are given in FIGS. 10 and 11, respectively. Since theresurfaced light chain of both antibodies included the CDR1 lysinesubstitution(s) for preferred version, a resurfaced version (v1.01) wasalso generated with murine lysine(s) retained in CDR-L1. Finally, heavychain framework 2 for murine EGFR-7 and its variants, contained asomatic W47C mutation resulting in an unpaired cysteine residue. Forthis reason, EGFR-7 heavy chain W47 germline revertant versions (v1.11)were also tested. FIG. 12 shows the alignment of the resurfacedsequences for EGFR-7 and EGFR-12 antibodies variable domain of bothlight and heavy chain with their murine counterparts.

TABLE 14 EGFR-7 CDR's (Resurfacing) EGFR-12 CDR's Light ChainLight Chain Murine and resurfaced v1.01 CDR1:Murine and resurfaced v1.01 CDR1: KASQDINNYLA (SEQ ID NO: 10)RASKSISKYLA (SEQ ID NO: 15) Resurfaced v1.0 CDR1: Resurfaced v1.0 CDR1:RASQDINNYLA (SEQ ID NO: 13) RASQSISRYLA (SEQ ID NO: 16) CDR2:  CDR2: YTSTLHP (SEQ ID NO: 11) SGSTLQS (SEQ ID NO: 17) CDR3:  CDR3: LQYDNLLYT (SEQ ID NO: 12) QQHNEYPWT (SEQ ID NO: 18) Heavy ChainHeavy Chain CDR1:  CDR1:  TSYWMQ (SEQ ID NO: 1) TSYWMQ (SEQ ID NO: 1)CDR2:  CDR2:  TIYPGDGDTT (SEQ ID NO: 2) TIYPGDGDTR (SEQ ID NO:7) CDR3: CDR3:  YDAPGYAMDY (SEQ ID NO: 3) YDAPGYAMDY (SEQ ID NO: 3)Kabat EGFR-7 HC CDR2 Kabat EGFR-12 HC CDR2 Murine HC CDR2:Murine HC CDR2: TIYPGDGDTTYTQKFKG (SEQ ID NO: 63)TIYPGDGDTRYIQKFKG (SEQ ID NO: 8) Humanized HC CDR2: Humanized HC CDR2:TIYPGDGDTTYTQKFQG (SEQ ID NO: 64) TIYPGDGDTRYIQKFQG (SEQ ID NO: 9)

In addition to humanization by variable domain resurfacing, EGFR-7antibody was also humanized following complementary determining region(CDR) grafting technology described, such as for example in Jones etal., Nature 321: 604-608 (1986) and Verhoeyen et al., Science 239:1534-1536 (1988). CDR grafting method consists of grafting the CDRs froma naturally evolved murine antibody onto the Fv framework regions (FRs)of a human antibody. The main step of the process was to choose theappropriate human acceptor frameworks. Kabat numbering scheme and KabatCDR definition were used for CDR grafting of EGFR-7 antibody. ExemplaryCDRs of EGFR-7 antibody for CDR grafting are defined as indicated inTable 15. Human immunoglobulin germline sequence with the highesthomology with the murine EGFR-7 antibody was identified through theinteractive tool, V-QUEST, of the International ImMunoGeneTicsinformation System® (IMGT (http://imgt.cines.fr/) as described inLefranc, Nucleic Acids Res. 29: 207-209 (2001). The human germlinesequences used as the acceptor frameworks for the VL and VH domains ofEGFR-7 antibody were IGKV1-33*01 and IGHV1-46*03, respectively. Tominimize concerns about the impact of conjugating lysines that fall inCDR's, lysine 24 in murine EGFR-7 antibody light chain CDR1 was replacedwith arginine in CDR grafting (Table 6). The specific framework residuechanges as well as the substitution in CDR-L1 in CDR-grafting of EGFR-7antibody are given in FIG. 13, and the alignments of the CDR-graftedsequences for EGFR-7 antibody variable domains with its murinecounterparts is illustrated in FIG. 14.

TABLE 15 EGFR-7 CDRs (CDR grafting) Light Chain Murine CDR1:KASQDINNYLA (SEQ ID NO: 10) CDR grafted CDR1:RASQDINNYLA (SEQ ID NO: 13) CDR2: YTSTLHP (SEQ ID NO: 11) CDR3:LQYDNLLYT (SEQ ID NO: 12) Heavy Chain CDR1: TSYWMQ (SEQ ID NO: 1) CDR2:TIYPGDGDTTYTQKFKG (SEQ ID NO: 63) CDR3: YDAPGYAMDY (SEQ ID NO: 3)

Recombinant Expression of the Humanized EGFR Antibodies

The variable region sequences for huEGFR-7 and huEGFR-12 werecodon-optimized and synthesized by Blue Heron Biotechnology. Thesequences were flanked by restriction enzyme sites for cloning in-framewith the respective constant sequences in single chain mammalianexpression plasmids. The light chain variable region was cloned intoEcoRI and BsiWI sites in the pAbKZeo plasmid. The heavy chain variableregion was cloned into the HindIII and Apa1 sites in the pAbG1Neoplasmid. These plasmids were used to express the recombinant antibodiesin either transient or stable mammalian cell transfections. Transienttransfections to express recombinant antibodies in HEK 293T cells wereperformed using a modified PEI procedure (Durocher, Y. et al., NucleicAcids Res. 30:E9 (2002)). Supernatant was purified by Protein A andpolishing chromatography steps using standard procedures as describedabove for murine antibodies.

Example 11 Binding Competition Between Murine and Humanized Anti-EGFRAntibodies

To examine the binding of humanized anti-EGFR antibodies, antibodycompetition assays were performed as described in Example 8. BecausemuEGFR-6 and muEGFR-7 antibodies cross-compete with one another (FIG.8C), this experiment examines the ability of murine or humanizedantibodies of EGFR-6 (FIG. 15A) or EGFR-7 (FIG. 15B) to compete with thebinding of biotinylated muEGFR-7 antibody to the MDA-MB468 cells. Inbrief, 1 nM of biotinylated muEGFR-7 antibody was pre-mixed with variousconcentration of ‘competing’ antibody. The antibody mixture was thenincubated with target cells on ice for 1 hour, washed, incubated withstreptavidin-APC conjugate on ice for another hour, washed, fixed andanalyzed using flow cytometer. The geometric mean fluorescence intensitywas plotted against antibody concentration in semi-log plot andnormalized so that 100% represents the maximal binding of thebiotinylated antibody in absence of other antibody and 0% represents thebackground staining in absence of the biotinylated antibody. As shown inFIG. 15A, both muEGFR-6 and huEGFR-6 antibodies compete the binding ofmuEGFR7 at similar EC50 (8.13 nM for muEGFR-6 and 11.09 nM forhuEGFR-6). FIG. 15B shows that muEGFR-7, hu-EGFR-7 and huEGFR-7Rantibodies also compete the muEGFR-7 antibody binding at similar EC50(3.54 nM for muEGFR7, 3.35 nM for huEGFR-7R and 4.12 nM for huEGFR-7).These results indicate that humanization does not affect the binding ofantibodies of the invention.

Anti-Tumor Activity of Humanized Anti-EGFR Antibodies

To examine the anti-tumor activity of the humanized antibodies of theinvention, H292 tumor cell growth inhibition assays were performed asdescribed in Example 7. As shown in FIG. 16, murine and humanizedantibodies of EGFR-6 and EGFR-7 were potent in inhibiting H292 tumorcell growth with EC50 included in Table 16. It is apparent that allhumanized antibodies maintain the anti-tumor activity of the murinecounterparts and humanization does not affect the biological activity ofthese antibodies.

TABLE 16 Anti-proliferative activity of anti- EGFR antibodies inNCI-H292 cells Antibody EC50 (nM) muEGFR-6 2.59 huEGFR-6 1.61 muEGFR-70.21 huEGFR-7 0.17 huEGFR-7R 0.11

Example 12 Antibody-Dependent-Cellular-Cytotoxicity (ADCC) Activity ofHuEGFR Antibodies

A lactate dehydrogenase (LDH) release assay was used to measureantibody-dependent cell mediated cytotoxicity (ADCC) of tumor cellslines using freshly isolated human natural killer (NK) cells as effectorcells (Shields R L, J Biol Chem. 2001 276(9):6591-604). The NK cellswere first isolated from human peripheral blood from a normal donor(Research Blood Components, Inc., Brighton, Mass.) using a modifiedprotocol for the NK cell Isolation Kit II (#130-091-152; MiltenyiBiotec, Auburn, Calif.). Peripheral blood was diluted 2-fold with 1×PBS.25 mL of diluted blood was carefully layered over 25 mL of Ficoll Paquein a 50 mL conical tube and centrifuged at 400 g for 45 min at RT. Theperipheral blood mononuclear cells (PBMC) were collected from theinterface, transferred into a new conical 50 mL tube, and washed oncewith 1×PBS. The PBMC were counted and resuspended at concentration of2.5×10⁷ cells/100 μl with MACS buffer (1×PBS, 0.5% BSA, 2 mM EDTA), andthen ¼× volume of NK cell Biotin-Antibody Cocktail were added to thecell suspension. The NK cell Biotin-Antibody Cocktail containsbiotinylated antibodies that bind to the lymphocytes, except for NKcells, resulting in a negative selection of NK cells. The mixture wasincubated at 4° C. for 10 min, and then ⅗× volume of MACS buffer and ⅖×volume of NK cell MicroBead cocktail that would bind to the biotinylatedantibodies were added. The cell-antibody mixture was incubated foranother 15 min at 4° C. Next, cells were washed once with 50 mL of MACSbuffer and resuspended in 3 mL of MACS buffer. NK cells were separatedas negative fraction using autoMACS separator (Miltenyi Biotec). Theresulting NK cells were plated into 30 mL of complete RPMI media(RPMI-1640 supplemented with 5% fetal bovine serum, 1%penicillin-streptomycin, 1 mM HEPES, 1 mM Sodium Pyruvate, 1% 100×MEMnon-essential Amino Acid Solution) overnight. The subsequent assay andall dilutions were carried out in RHBP medium (RPMI-1640 mediumsupplemented with 20 mM HEPES, pH 7.4, 0.1% BSA and 1%penicillin-streptomycin).

Various concentrations of antibodies in RHBP medium were aliquoted induplicate at 50 μL/well into a round bottom 96-well plate. The targetcells (in this experiment A431 cell line) were resuspended at 10⁶cells/mL in RHBP medium and added at 100 μL/well to each well containingantibody dilutions. The plate containing target cells and antibodydilutions was incubated for 30 min at RT. NK cells were then added tothe wells containing the target cells at 50 μL/well. The typical ratiowas 1 target cell to 3-4 NK cells. The following controls were set upfor each experiment: NK cells alone, target cells alone (spontaneous LDHrelease), target cells with NK cells (antibody independent LDH release),target cells with 10% Triton X-100 (maximum LDH release). The mixtureswere incubated at 37° C. for 4 h to allow for cell lysis. Plates werecentrifuged for 10 min at 1200 rpm, and 100 μL of the supernatant wascarefully transferred to a new flat-bottom 96-well plate. LDH reactionmixture (100 μL/well) from the Cytotoxicity Detection Kit (Roche 1 644793) was added to each well and incubated at room temperature for 5 to30 min. The optical density (OD) of samples was measured at 490 nm(OD490). The percent specific lysis of each sample was determined usingthe following formula: percent specific lysis=(sample value−spontaneousrelease)/(maximum release−spontaneous release)*100.

FIG. 17 shows a representative ADCC activity of huEGFR-6 and huEGFR-7Rantibodies in comparison to that of chKTI antibody. The huEGFR-6 andhuEGFR-7R antibodies induced NK cell mediated killing of target cells indose dependent manner with maximal specific killing reached around 20%and EC50 of 22 ng/ml and 17 ng/ml for huEGFR6 and huEGFR-7R,respectively. In contrast, chKTI antibody that did not bind to targetcells failed to mediate ADCC.

Example 13 Preparation of huEGFR-7R-SMCC-DM1

The (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,Pierce Biotechnology, Inc) linker was dissolved in dimethylacetamide(DMA). The huEGFR antibody was modified with SMCC to introducemaleimides into the antibody by incubating the antibody at 5 mg/mL in 50mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5 with a 10 molarexcess of SMCC. After approximately 100 minutes at ambient temperature,the reaction mixture was purified using a SEPHADEX™ G25 columnequilibrated with the same potassium phosphate buffer. Antibodycontaining fractions were pooled and used for subsequent steps.

The SMCC-modified antibody was reacted with a 10 mM solution of DM1 at a1.7 molar excess relative to the maleimide linker. The reaction wasstirred at ambient temperature under for approximately 18 hours. Theconjugation reaction mixture was filtered through a SEPHADEX™ G25 gelfiltration column equilibrated with 1×PBS at pH 6.5. The huEGFRantibody-SMCC-DM1 conjugate was then dialyzed into buffer containing 10mM histidine, 250 mM glycine, 1% sucrose pH 5.5. The number of DM1molecules linked per antibody molecule was determined using thepreviously reported extinction coefficients for antibody and DM1 (Liu etal., Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). The percentageof free maytansinoid present after the conjugation reaction wasdetermined by injecting 20-50 μg conjugate onto a HiSep columnequilibrated in 25% acetonitrile in 100 mM ammonium acetate buffer, pH7.0, and eluting in acetonitrile. The peak area of total freemaytansinoid species (eluted in the gradient and identified bycomparison of elution time with known standards) was measured using anabsorbance detector set to a wavelength of 252 nm and compared with thepeak area related to bound maytansinoid (eluted in the conjugate peak inthe column flow-through fractions) to calculate the percentage of totalfree maytansinoid species. Conjugates with 3.5-4 DM1 molecules perhuEGFR antibody were obtained with <1% present as unconjugatedmaytansinoid.

Preparation of huEGFR-7R-SPDB-DM4

The exemplary N-succinimidyl 4-(2-pyridyldithio) butanoate (SPDB) linkerwas dissolved in ethanol. The huEGFR antibody was incubated at 8 mg/mLwith a 5.5-5 fold molar excess of SPDB linker for approximately 2 hoursat room temperature in 50 mM potassium phosphate buffer (pH 6.5)containing 50 mM NaCl, 2 mM EDTA, and 3% ethanol. The SPDB modifiedantibody was diluted 2-fold in PBS, pH 6.5 and modified with a 1.5 foldmolar excess of the maytansinoid DM4 by the addition of a concentratedsolution (15-30 mM) of DM4 in dimethylacetamide (DMA). After overnightincubation at room temperature, the conjugated antibody was purified bychromatography on SEPHADEX™ G25F equilibrated with 10 mM histidine, 250mM glycine, 1% sucrose pH 5.5. The number of DM4 molecules linked perantibody molecule was determined using the previously reportedextinction coefficients for antibody and maytansinoid (Widdison W C etal. J Med Chem, 49:4392-4408 (2006)). The percentage of total freemaytansinoid species were determined as described above. Conjugates with3.5-4 DM4 molecules per huEGFR antibody were obtained with <1% presentas unconjugated maytansinoid.

Example 14 Binding Affinity of Maytansinoid Conjugates

Binding affinity of the huEGFR-6 and huEGFR-7R antibody maytansinoidconjugates was compared with that of the naked antibodies usingMDA-MB468 cells as described in the Example 2. The Kds calculated frombinding curves of the huEGFR-6 antibody and conjugates (FIG. 18 A) were0.81 nM for naked huEGFR-6 antibody and 1.18 nM for huEGFR-6-SMCC-DM1conjugate. The Kds calculated from binding curves of the huEGFR-7Rantibody and conjugates (FIG. 18 B) were 0.67 nM for naked huEGFR-7Rantibody and 0.83 nM for huEGFR-7R-SMCC-DM1 conjugate. This datademonstrates that DM conjugation does not notably alter the bindingaffinity of the huEGFR-6 and huEGFR-7R antibody to the huEGFR antigen.

Example 15 In Vitro Cytotoxic Assay on Tumor Cells

The ability of EGFR antibody maytansinoid conjugates to inhibit thetumor cell growth was measured using in vitro cytotoxicity assays.Briefly, target cells were plated at 1,500 to 3,000 cells per well in100 μL complete RPMI media containing 10% FBS. Conjugates were dilutedinto complete RPMI media using 5-fold dilution series and 100 μL wereadded per well. The final concentration typically ranged from 3×10⁻⁸ Mto 8×10⁻¹⁴ M. Cells were incubated at 37° C. in a humidified 5% CO2incubator for 5 days. Viability of the remaining cells was determined bycolorimetric WST-8 assay and the absorbance at 450 nm (A450) wasmeasured in a multiwell plate reader. The surviving fraction wascalculated by dividing each treated sample value by the average value ofuntreated controls. The surviving fraction value was plotted against theantibody-conjugate concentration in a semi-log plot for each treatment.

The in vitro cytotoxicity of naked antibodies and antibody-maytansinoidconjugates of the invention was compared to the activity of anon-specific antibody and its corresponding maytansinoid conjugate suchas chKTI and chKTI-SMCC-DM1. The results from a typical cytotoxicityassay are shown in FIGS. 19 and 20.

In FIG. 19A, the activity of naked antibodies and maytansinoidconjugates of the invention was tested in FaDu cell line. The huEGFR-6and huEGFR-7R naked antibodies inhibited 40% and 55% H292 cell growth,respectively while the chKTI antibody had no activity. Maytansinoidconjugation further enhances the activity of the EGFR antibody of theinvention. Both huEGFR-6 and huEGFR-7R-SMCC-DM1 conjugates completelyabolished the target cells with EC50 of 0.22 nM and 0.06 nM,respectively. The control chKTI-SMCC-DM1 conjugate also killed thetarget cells but with much lower EC50 (0.59 μM).

In FIG. 19B, the activity of naked antibodies and maytansinoidconjugates of the invention was tested in H292 cell line. The huEGFR-6and huEGFR-7R naked antibodies inhibited 60-70% H292 cell growth whilethe chKTI antibody had no activity. Maytansinoid conjugation furtherenhances the activity of the EGFR antibody of the invention. BothhuEGFR-6 and huEGFR-7R-SMCC-DM1 conjugates completely abolished thetarget cells with EC50 of 0.16 nM and 0.03 nM, respectively. The controlchKTI-SMCC-DM1 conjugate also killed the target cells but with muchlower EC50 (38.51 nM).

In FIG. 20A, the activity of naked antibodies and maytansinoidconjugates of the invention was compared with cetuximab in H226 cellline. The cetuximab, huEGFR-6, huEGFR-7R naked antibodies as well as thechKTI control antibody had no anti-proliferative activity. Maytansinoidconjugates of both huEGFR-6 and huEGFR-7R antibodies completelyeliminated the target cells with EC50 of 0.68 nM and 0.14 nM,respectively while the control chKTI-SMCC-DM1 conjugate failed to killthe target cells.

In FIG. 20B, the activity of naked antibodies and maytansinoidconjugates of the invention was compared with cetuximab in the SCC-4cell line. The cetuximab, huEGFR-6 and huEGFR-7R naked antibodies showeda dose dependent growth inhibition with maximal inhibition of around30%, while the chKTI antibody had no activity. Maytansinoid conjugationfurther potentiates the activity of the EGFR antibody of the invention.Both huEGFR-6 and huEGFR-7R-SMCC-DM1 conjugates completely eliminatedthe target cells with EC50 of 0.07 nM and 0.03 nM, respectively. Thecontrol chKTI-SMCC-DM1 conjugate also killed the target cells but with amuch lower EC50 (17.62 nM). Altogether, these results show that themaytansinoid conjugation dramatically enhances the anti-tumor activityof the EGFR antibodies of the invention.

Example 16 In Vivo Efficacy Study Comparing HuEGFR-6 and HuEGFR-7RAntibodies and Maytansinoid Conjugates

The activity of naked antibody and antibody-maytansinoid conjugates ofthe huEGFR-6 and huEGFR-7R antibodies was tested in EGFR expressing H292NSCLC (non-small cell lung cancer) (FIG. 21) and FaDu SCCHN (squamouscell carcinoma of head and neck) (FIG. 22) tumor xenograft models. 1×10⁷tumor cells were injected subcutaneously into SCID mice. Animals wererandomized by tumor volume into treatment groups when tumors reached amean tumor volume of approximately 100 mm³ and injected once with theindicated dosage of naked antibodies or antibody-maytansinoidconjugates. Median tumor volume of each treatment groups is plottedagainst time post tumor cell inoculation (FIGS. 21 and 22). Tables 17and 18 show the number of mice with complete response (CR) (no palpabletumor) and percent of tumor growth inhibition (% T/C) which correspondsto the median of tumor volume of each treated group divided by themedian tumor volume of control group when the tumor volume of thecontrol group reaches a predetermined size. A treatment with a % T/Cvalue of below 42% is considered active, while a treatment with a % T/Cvalue of below 12% is considered highly active.

Both the naked antibodies and antibody maytansinoid conjugates of theinvention were very active and they significantly delayed the growth ofboth H292 and FaDu tumor xenografts (FIGS. 21 and 22). In H292 tumorxenograft study (FIG. 21 and Table 17), all mice treated with thehuEGFR-6-SMCC-DM1 and huEGFR-7R-SMCC-DM1 conjugates as low as 3 mg/kgexhibited a complete response. Even the huEGFR-6 and huEGFR-7Rantibodies at 10 mg/kg were highly active. In FaDu tumor xenograft study(FIG. 22 and Table 18), the huEGFR-6 and the huEGFR-7R antibodies at 10mg/kg were active and the antibody-maytansinoid conjugates were evenmore active with complete response in some mice.

TABLE 17 Activity of EGFR Ab and maytansinoid conjugates in H292 tumorxenograft Ab and conjugate % T/C CR huEGFR-6 Ab 10 mg/kg 3.1 1/6huEGFR-6-SMCC-DM1 3 mg/kg 0.0 6/6 huEGFR-6-SMCC-DM1 10 mg/kg 0.0 6/6huEGFR-7R Ab 10 mg/kg 2.2 4/6 huEGFR-7R-SMCC-DM1 3 mg/kg 1.2 6/6huEGFR-7R-SMCC-DM1 10 mg/kg 0.6 6/6

TABLE 18 Activity of EGFR Ab and maytansinoid conjugates in FaDu tumorxenograft Ab and conjugate % T/C CR huEGFR-6 Ab 10 mg/kg 17.5 0/6huEGFR-6-SMCC-DM1 10 mg/kg 5.8 2/6 huEGFR-7R Ab 10 mg/kg 13.4 0/6huEGFR-7R-SMCC-DM1 10 mg/kg 5.5 1/6In Vivo Efficacy Study Comparing huEGFR-7R-SMCC-DM1 andhuEGFR-7R-PEG-MAL-DM1

The activity of naked antibody, the huEGFR-7R-SMCC-DM1 and thehuEGFR-7R-PEG-MAL-DM1 conjugates was compared in EGFR expressing H292NSCLC (non-small cell lung cancer) (FIG. 25), HSC2 SCCHN (squamous cellcarcinoma of head and neck) (FIG. 26) and FaDu SCCHN (FIG. 27) tumorxenograft models. The experiment and data analysis were done asdescribed above.

In H292 NSCLC tumor xenograft study (FIG. 25 and Table 19), all of thetest articles were highly active at 3 mg/kg single dose. All micetreated with both the huEGFR-7R-SMCC-DM1 and the huEGFR-7R-PEG-MAL-DM1conjugates showed a complete response, while none of the mice treatedwith the huEGFR-7R antibody had a complete response. In HSC2 SCCHN tumorxenograft study (FIG. 26 and Table 20), both the huEGFR-7R-SMCC-DM1 andthe huEGFR-7R-PEG-MAL-DM1 conjugates were highly active with T/C of 8%,while the huEGFR-7R antibody was barely active at 5 mg/kg single dose.In FaDu SCCHN tumor xenograft study (FIG. 27 and Table 21), both thehuEGFR-7R-SMCC-DM1 and the huEGFR-7R-PEG-MAL-DM1 conjugates were activewith T/C of 15% and 28%, respectively. The huEGFR-7R antibody treatmentshowed some tumor growth inhibition but it was not significantly active.In conclusion, these results show that the naked antibodies of theinvention are potent in inhibiting the growth of NSCLC and SCCHN tumors,and the conjugation with maytansinoid further enhances the anti-tumoractivity.

TABLE 19 Activity of huEGFR-7R Ab and maytansinoid conjugates in H292tumor xenograft Ab and conjugate % T/C CR huEGFR-7R Ab 3 mg/kg 10 0/6huEGFR-7R-SMCC-DM1 3 mg/kg <1 6/6 huEGFR-7R-PEG-MAL-DM1 3 mg/kg <1 6/6

TABLE 20 Activity of huEGFR-7R Ab and maytansinoid conjugates in HSC2tumor xenograft Ab and conjugate % T/C CR huEGFR-7R Ab 5 mg/kg 41 0/6huEGFR-7R-SMCC-DM1 5 mg/kg 8 0/6 huEGFR-7R-PEG-MAL-DM1 5 mg/kg 8 0/6

TABLE 21 Activity of huEGFR-7R Ab and maytansinoid conjugates in FaDutumor xenograft Ab and conjugate % T/C CR huEGFR-7R Ab 5 mg/kg 45 0/6huEGFR-7R-SMCC-DM1 5 mg/kg 15 0/6 huEGFR-7R-PEG-MAL-DM1 5 mg/kg 28 0/6

Example 17 In Vitro Cytotoxicity Assay on Human Primary Keratinocytes

EGFR signaling plays a key role in human primary keratinocyteproliferation Inhibition of EGFR signaling by small molecule tyrosinekinase inhibitors or antagonistic anti-EGFR antibodies such as cetuximableads to growth arrest and apoptosis in keratinocyte culture (Stoll etal., Oncogene 16, 1493-1499 (1998)). Keratinocyte apoptosis is thoughtto be one of the mechanism underlying dermatologic toxicities caused bythe anti-EGFR therapies in the clinic. To examine the potential of skintoxicity, the EGFR antibodies and antibody-maytansinoid conjugates ofthe invention was tested in an in vitro cytotoxicity assay using humanprimary keratinocytes. Briefly, human primary keratinocytes (Invitrogen)were plated at 1,500 to 3,000 cells per well in 100 μL EGF containingmedia suggested by the manufacturer. Test articles were diluted in EGFcontaining media using 5-fold dilution series and 100 μL were added perwell. The final concentration typically ranged from 3×10⁻⁸ M to 8×10⁻¹⁴M. Cells were incubated at 37° C. in a humidified 5% CO2 incubator for 5days. Viability of the remaining cells was determined by colorimetricWST-8 assay and the absorbance at 450 nm (A450) was measured in amultiwell plate reader. The surviving fraction was calculated bydividing each treated sample value by the average value of untreatedcontrols. The surviving fraction value was plotted against theantibody-conjugate concentration in a semi-log plot for each treatment.

The in vitro cytotoxicity of huEGFR-7R naked antibodies andhuEGFR-7R-SMCC-DM1 conjugate was compared to the activity of anon-specific antibody (chKTI), chKTI-SMCC-DM1 conjugate, a nonantagonistic antibody (huML66) and its corresponding maytansinoidconjugate on the human primary keratinocytes (FIG. 23A) as well as H292tumor cells (FIG. 23B) in the same experiment. In H292 cell line (FIG.23B), huML66 antibody had no activity, while the huEGFR-7R antibodyinhibited cell growth up to 55%. The maytansinoid conjugates of huML66and huEGFR-7R had the best activity; they were able to completelyinhibit tumor cell growth with EC50 between 0.05 and 0.07 nM. In humanprimary keratinocytes (FIG. 23A), chKTI and huML66 antibodies had noeffect on keratinocyte proliferation. However, the huML66-SMCC-DM1 wasvery potent in killing the keratinocytes with 0.55 nM EC50. ThehuEGFR-7R naked antibody had very little effect on keratinocytes.Surprisingly, the huEGFR-7R-SMCC-DM1 conjugate was much less toxic tothe keratinocytes as compared to the huML66-SMCC-DM1 conjugate. At theconcentration of 3.3 nM, the huEGFR-7R naked antibody and itscorresponding conjugate only inhibited less than 40% of the keratinocytegrowth. In summary, antibody and antibody-maytansinoid conjugate of theinvention has little effect on the human primary keratinocyte cellgrowth while they are very potent in eliminating tumor cells.

Example 18 Chemokine/Cytokine Production by Human Primary Keratinocytes

The skin epithelium, which is composed mainly of keratinocytesinterspersed with dendritic cells, melanocytes, and rare T lymphocytesand monocytes, is highly committed to host defense. Physical, chemical,or immune-specific insults rapidly evoke an epidermal responsecharacterized by the increase expression of chemokine and cytokines,which attract and activate distinct leukocyte subpopulations to induceinflammatory response. TNFα induces human keratinocytes to expressnumerous chemokines and cytokines including CCL5/RANTES,CXCL10/IFNγ-inducible-protein 10 and CXCL8/IL-8. CCL5 attracts T cells,monocytes as well as neutrophils. CXCL10 induces migration of type 1 Tcells. CXCL8 is a chemoattractants active in neutrophil recruitment aswell as in epithelial and endothelial cell proliferation.

EGFR signaling governs the homeostatic maintenance and repair ofepithelial tissue. EGFR activation leads to keratinocyte proliferation,migration and controlled differentiation. In response to TNFα,keratinocytes produce EGFR ligands which activate EGFR signaling. Theenhanced EGFR activation in keratinocytes increases CXCL8 expression andreduces CCL5 and CXCL10 expression. In contrast, impairment of EGFRsignaling led to an opposite pattern Skin application of a selectiveEGFR tyrosine kinase inhibitor led to more severe contacthypersensitivity responses, with increased epidermal levels of CCL5 andCXCL10, and a higher number of monocytes/macrophages and T cells in theskin. These findings suggested that EGFR signaling modulates skininflammation by affecting chemokine expression in keratinocytes(Pastore, J. Immunol., 174: 5047-5056 (2005)). It is now believed thatskin toxicity manifested in the clinic upon EGFR therapies are caused byapoptosis and sustained inflammation in the skin.

The effect of antibodies and antibody-maytansinoid conjugates of theinvention in modulating chemokine/cytokine production by human primarykeratinocytes was tested in the following in vitro assay. 1×10⁵ humanprimary keratinocytes/well (Invitrogen) were first seeded in 6 wellplate. The cells were starved overnight and then cultured with 100 ng/mlTNFα and 10 μg/ml test antibodies in the EGF containing media for 14hours. The amount of CCL5, CXCL10 and CXCL8 in the culture supernatantwas measured using ELISA kits from R&D systems according to themanufacturer's protocol. As shown in FIG. 24, cetuximab reduced theexpression of CXCL8 and increased the production of CXCL10 and CCL5. Incontrast, the naked antibodies of the invention had no or little effecton the expression of these chemokines/cytokines when compared to thechKTI control. Surprisingly, the maytansinoid conjugates of the EGFRantibodies of the invention also had no or little effect on thekeratinocytes. Altogether, the results shown in Examples 17 and 18strongly suggest that both the antibodies and antibody maytansinoidconjugates of the invention had minimal effect on the human primarykeratinocytes in vitro, therefore are likely to be less toxic to theskin in humans. In contrast to the effect on keratinocytes, theantibodies and antibody maytansinoid conjugates of the invention arevery potent in killing the EGFR positive tumor cells in vitro and invivo as shown in Examples 7, 12, 15 and 16. In summary, the antibodiesand antibody cytotoxic agent conjugates of the invention are uniqueclass of anti-EGFR molecules that have distinct effect on normal vs.tumor cells.

Example 19 Epitope Mapping

The human EGFR is a large (1186 residues), monomeric glycoprotein withan extracellular ligand binding region, a single transmembrane regionand a cytoplasmic tyrosine kinase domain flanked by noncatalyticregulatory regions. The extracellular domain (ECD) of human EGFR(residues 1-618) contains four subdomains (FIG. 28), here termed domainI (amino acids 1-165), domain II (amino acids 166-309), domain III(amino acids 310-481), and domain IV (amino acids 482-618). Thesedomains are also referred to as L1, CR1, L2, and CR2, where L and CR areacronyms for large and Cys-rich, respectively. The epitope of thehuEGFR-7R of the invention were mapped mainly to the defined regioncontaining amino acids 460-480 in the human EGFR ECD domain III byengineering truncated and chimeric human/murine EGFR molecules.

EGFR Variants Cloning and Expression

The entire human EGFR ECD (amino acids 1-618) was expressed as an Fcfusion protein (huEGFR-Fc). The protein sequence was codon optimized,synthesized, and cloned in frame with a murine IgG2A hinge, CH2, and CH3region in the pmuFc2ANL mammalian expression vector by Blue HeronBiotechnologies. As antibodies of the invention compete with cetuximabbinding to EGFR (FIG. 8B), and cetuximab binds exclusively to domainIII, the epitope of the antibodies of the invention may also be locatedin domain III and might overlap with that of cetuximab. To furtheridentify the epitope, an Fc fusion of the truncated human EGFR(huEGFRdIII-Fc), containing entire domain III (amino acids 310-481) plus20 extra residues from domain IV (amino acids 482-501), which wassuggested to be required for binding of cetuximab, was similarlyconstructed as huEGFR-Fc. Further, a truncated murine EGFR containingamino acids 310-501 (muEGFRdIII-Fc), and chEGFRdIII-Fc, a chimericversion containing murine EGFR amino acids 310-501 with nucleotidesequence coding for amino acids 460-481 being replaced by thecorresponding sequence from human EGFR, were also similarly constructedto be expressed as Fc fusion proteins (FIG. 29). The chEGFRdIII-Fcconstructs consists of 10 amino acids mutations to their humancounterparts, including residues 460, 461, 467, 468, 471, 473, 474, and478-480. All forms of EGFR ECD Fc tagged proteins were expressed viatransient transfection of HEK 293T cells and purified from thesupernatant of the transfected cells using protein A affinitychromatography.

Antibody Binding to Various EGFR ECD-Fc Constructs

The huEGFR-7R was tested in ELISA format for binding to the variousEGFR-Fc constructs described above. As shown in FIG. 30, huEGFR-7Rantibody binds to both human EGFR (huEGFR-Fc) and human EGFR domain III(huEGFRdIII-Fc) with similar affinity. FIG. 30 also demonstrates thathuEGFR-7R antibody practically does not recognize the murine EGFR domainIII (muEGFRdIII-Fc), despite the high sequence homology with the humanreceptor (88% sequence identity in domain III). Additionally, thehuEGFR-7R antibody binds to the human/murine EGFR chimera(chEGFRdIII-Fc), containing mainly murine EGFR domain III sequence withten amino acids at positions 460, 461, 467, 468, 471, 473, 474, and478-480 mutated to their human counterparts (FIG. 30). These dataindicate that huEGFR-7R antibody binds exclusively to the domain III ofhuman EGFR and the binding epitope is largely confined within amino acidpositions 460-480. When the binding affinities to different truncatedformats of huEGFR are compared, it is apparent that there was anapproximately two fold decrease in the binding affinity of the huEGFR-7Rantibody to chEGFRdIII-Fc as compared to huEGFR and huEGFRdIII,suggesting that the epitope of huEGFR-7R antibody consists of additionalamino acid residues besides those in the positions 460-480. This datawas confirmed with the muEGFR-7 antibody binding results (FIG. 32).

In parallel, other anti-EGFR antibodies of the invention, such ashuEGFR-6 (FIG. 31), muEGFR-6 (FIG. 33), muEGFR-12 (FIG. 34) andmuEGFR-13 (FIG. 35) antibodies that share unique biological activitieswith the EGFR-7 antibody, exhibit similar binding properties as theEGFR-7 antibody. They bind to human EGFR domain III (huEGFRdIII-Fc) butnot murine EGFR domain III (muEGFRdIII-Fc), and importantly, they allbind to the chEGFRdIII-Fc at a lower affinity than to the huEGFRdIII-Fc.These data suggest that the epitope recognized by the antibodies of theinvention constitutes other residues in domain III in addition to theamino acid residues in positions 460-480. In contrast, cetuximab bindsto chEGFRdIII as well as wild type human EGFR and huEGFRdIII at similaraffinity (FIG. 36), suggesting that the cetuximab binding epitope isconfined to amino acid residues in positions 460-480. In summary, thehuEGFR-7R antibody along with other anti-EGFR antibodies of theinvention binds exclusively to domain III of the huEGFR extracellulardomain. Moreover, through constructing chimeric EGFR, it has beenconfirmed that the epitope recognized by the antibodies of the inventionis displaced toward the C-terminus of huEGFR domain III and largelyoverlaps with, but is not identical, to the cetuximab binding site andvery likely consists of additional critical amino acids

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

1. An antibody or antigen binding fragment thereof that specificallybinds to human EGFR, wherein said antibody has at least onecharacteristic selected from the group consisting of: (a) inhibits atleast 80% of epidermal growth factor (EGF) and transforming growthfactor alpha (TGFα) binding to A431 cells at a concentration of 10 nM orhigher, (b) causes at least 50% inhibition of H292 and HCC827 tumor cellproliferation at 30 nM or higher, and (c) does not inhibit more than 20%proliferation of keratinocytes and MCF-10A epithelial cells at 60 nM orlower. 2-3. (canceled)
 4. The antibody or antigen binding fragmentthereof of claim 1, wherein the antibody comprises (a) a VH sequence atleast 90%, at least about 95%, at least about 99%, or 100% identical toa reference VH sequence selected from the group consisting of SEQ IDNOs:19-23, 69, and 71-73; and (b) a VL sequence at least 90%, at leastabout 95%, at least about 99%, or 100% identical to a reference VLsequence selected from the group consisting of SEQ ID NOs:24-30 and 70.5-21. (canceled)
 22. (canceled)
 23. (canceled)
 24. An antibody orantigen binding fragment thereof that specifically binds to the sameEGFR epitope as an antibody selected from the group consisting of ATCCDeposit Designation PTA-11331, deposited with the ATCC on Oct. 6, 2010,ATCC Deposit Designation PTA-11332, deposited with the ATCC on Oct. 6,2010, and ATCC Deposit Designation PTA-11333, deposited with the ATCC onOct. 6,
 2010. 25. An antibody or antigen binding fragment thereof thatcompetitively inhibits binding of a reference antibody to human EGFR,wherein said reference antibody is selected from the group consisting ofATCC Deposit Designation PTA-11331, deposited with the ATCC on Oct. 6,2010, ATCC Deposit Designation PTA-11332, deposited with the ATCC onOct. 6, 2010, and ATCC Deposit Designation PTA-11333, deposited with theATCC on Oct. 6, 2010, and wherein the antibody or antigen bindingfragment: (a) inhibits at least 80% of epidermal growth factor (EGF) andtransforming growth factor alpha (TGFα) binding to A431 cells at aconcentration of 10 nM or higher, (b) causes at least 50% inhibition ofH292 and HCC827 tumor cell proliferation at 30 nM or higher, or (c) doesnot inhibit more than 20% proliferation of keratinocytes and MCF-10Aepithelial cells at 60 nM or lower.
 26. An antibody or antigen bindingfragment thereof that specifically binds to the same EGFR epitope, orcompetitively inhibits the binding of an antibody to human EGFR, as anantibody selected from the group consisting of: a) an antibodycomprising the VH polypeptide of SEQ ID NO:19 and the VL polypeptide ofSEQ ID NO:24; b) an antibody comprising the VH polypeptide of SEQ IDNO:20 and the VL polypeptide of SEQ ID NO:25; c) an antibody comprisingthe VH polypeptide of SEQ ID NO:21 and the VL polypeptide of SEQ IDNO:26; d) an antibody comprising the VH polypeptide of SEQ ID NO:21 andthe VL polypeptide of SEQ ID NO:27; e) an antibody comprising the VHpolypeptide of SEQ ID NO:22 and the VL polypeptide of SEQ ID NO:28; f)an antibody comprising the VH polypeptide of SEQ ID NO:23 and the VLpolypeptide of SEQ ID NO:29; g) an antibody comprising the VHpolypeptide of SEQ ID NO:23 and the VL polypeptide of SEQ ID NO:30; h)an antibody comprising the VH polypeptide of SEQ ID NO:69 and the VLpolypeptide of SEQ ID NO:70; i) an antibody comprising the VHpolypeptide of SEQ ID NO:71 and the VL polypeptide of SEQ ID NO:26; j)an antibody comprising the VH polypeptide of SEQ ID NO:71 and the VLpolypeptide of SEQ ID NO:27; k) an antibody comprising the VHpolypeptide of SEQ ID NO:72 and the VL polypeptide of SEQ ID NO:26; l)an antibody comprising the VH polypeptide of SEQ ID NO:72 and the VLpolypeptide of SEQ ID NO:27; m) an antibody comprising the VHpolypeptide of SEQ ID NO:73 and the VL polypeptide of SEQ ID NO:26; andn) an antibody comprising the VH polypeptide of SEQ ID NO:73 and the VLpolypeptide of SEQ ID NO:27.
 27. (canceled)
 28. An antibody or antigenbinding fragment thereof that specifically binds to human EGFR, whereinthe antibody or antigen binding fragment thereof comprises: a) animmunoglobulin heavy chain variable region comprising CDR1, CDR2, andCDR3, which are respectively identical to, or contain 1, 2, or 3conservative amino acid substitutions compared to the reference heavychain CDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2sequence of SEQ ID NO: 2, 4, 6, 63, or 64, and the reference heavy chainCDR3 sequence of SEQ ID NO: 3 or 5; and b) an immunoglobulin a lightchain variable region comprising CDR1, CDR2, and CDR3, which arerespectively identical to, or contain 1, 2, or 3 conservative amino acidsubstitutions compared to the reference light chain CDR1 sequence of SEQID NO: 10, 13, or 14, the reference light chain sequence CDR2 of SEQ IDNO:11, and the reference light chain CDR3 sequence of SEQ ID NO: 12; orc) an immunoglobulin heavy chain variable region comprising CDR1, CDR2,and CDR3, which are respectively identical to, or contain 1, 2, or 3conservative amino acid substitutions compared to the reference heavychain CDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2sequence of SEQ ID NO: 7, 8, or 9, and the reference heavy chain CDR3sequence of SEQ ID NO: 3; and d) an immunoglobulin a light chainvariable region comprising CDR1, CDR2, and CDR3, which, with theexception of 1, 2, or 3 conservative amino acid substitutions, arerespectively identical to, or contain 1, 2, or 3 conservative amino acidsubstitutions compared to the reference light chain CDR1 sequence of SEQID NO: 15 or 16, the reference light chain CDR2 sequence of SEQ IDNO:17, and the reference light chain CDR3 sequence of SEQ ID NO: 18; ore) an immunoglobulin heavy chain variable region comprising CDR1, CDR2,and CDR3, which are respectively identical to, or contain 1, 2, or 3conservative amino acid substitutions compared to the reference heavychain CDR1 sequence of SEQ ID NO:1, the reference heavy chain CDR2sequence of SEQ ID NO: 65, 66, or 67, and the reference heavy chain CDR3sequence of SEQ ID NO: 3; and f) an immunoglobulin a light chainvariable region comprising CDR1, CDR2, and CDR3, which are respectivelyidentical to, or contain 1, 2, or 3 conservative amino acidsubstitutions compared to the reference light chain CDR1 sequence of SEQID NO: 68 or 13, the reference light chain sequence CDR2 of SEQ IDNO:11, and the reference light chain CDR3 sequence of SEQ: ID NO: 12.29-42. (canceled)
 43. An isolated cell producing the antibody or antigenbinding fragment thereof of claim
 1. 44. A method of making the antibodyor antigen-binding fragment thereof of claim 1, comprising (a) culturingthe cell of claim 43; and (b) isolating said antibody, antigen-bindingfragment thereof from said cultured cell.
 45. (canceled)
 46. Animmunoconjugate having the formula (A)-(L)-(C), wherein: (A) is anantibody or antigen binding fragment thereof of claim 1; (L) is alinker; and (C) is a cytotoxic agent; and wherein said linker (L) links(A) to (C). 47-53. (canceled)
 54. A pharmaceutical compositioncomprising the antibody or antigen binding fragment thereof of claim 1and a pharmaceutically acceptable carrier.
 56. A diagnostic reagentcomprising the antibody or antigen binding fragment thereof of claim 1which is labeled.
 57. (canceled)
 58. A kit comprising the antibody orantigen binding fragment thereof of claim
 1. 59. A method for inhibitingthe growth of a cell expressing EGFR comprising contacting the cell withthe antibody or antigen binding fragment thereof, or polypeptide ofclaim
 1. 60. (canceled)
 61. A method for treating a patient havingcancer comprising administering to said patient a therapeuticallyeffective amount of the antibody or antigen binding fragment thereof ofclaim 1 to the patient. 62-63. (canceled)
 64. A method for treating acell proliferative disorder comprising administering to said patient atherapeutically effective amount of the antibody or antigen bindingfragment thereof of claim 1 to the patient.
 65. (canceled)
 66. Anisolated polynucleotide comprising a sequence that encodes a polypeptideat least 90%, at least 95%, at least 99%, or 100% identical to asequence selected from the group consisting of SEQ ID NOs: 39-50 and77-84. 67-72. (canceled)
 73. A vector comprising the polynucleotide ofclaim
 66. 74. A host cell comprising the vector of claim
 73. 75.(canceled)
 76. The immunoconjugate of claim 46, having the formula(A)-(L)-(C), wherein: (A) is an antibody or antigen binding fragmentthereof comprising the heavy chain variable region of SEQ ID NO:21 andthe light chain variable region of SEQ ID NOs: 26 or 27; (L) is aN-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) linker;and (C) is the cytotoxic agentN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1).
 77. Amethod for inhibiting the growth of a cell expressing EGFR comprisingcontacting the cell with the immunoconjugate of claim
 46. 78. A methodfor treating a patient having cancer comprising administering to saidpatient a therapeutically effective amount of the immunoconjugate ofclaim 46 to the patient.
 79. A method for treating a cell proliferativedisorder comprising administering to said patient a therapeuticallyeffective amount of the immunoconjugate of claim 46 to the patient. 80.An antibody or antigen binding fragment thereof of claim 26, thatcompetitively inhibits binding of a second antibody to human EGFR,wherein said second antibody comprises the heavy chain variable regionof SEQ ID NO:21 and the light chain variable region of SEQ ID NOs: 26 or27, and wherein the first antibody or antigen binding fragment: (a)inhibits at least 80% of epidermal growth factor (EGF) and transforminggrowth factor alpha (TGFα) binding to A431 cells at a concentration of10 nM or higher, (b) causes at least 50% inhibition of H292 and HCC827tumor cell proliferation at 30 nM or higher, or (c) does not inhibitmore than 20% proliferation of keratinocytes and MCF-10A epithelialcells at 60 nM or lower.
 81. A polypeptide, wherein the polypeptidecomprises (a) a VH sequence at least 90%, at least about 95%, at leastabout 99%, or 100% identical to a reference VH sequence selected fromthe group consisting of SEQ ID NOs:19-23, 69, and 71-73; and (b) a VLsequence at least 90%, at least about 95%, at least about 99%, or 100%identical to a reference VL sequence selected from the group consistingof SEQ ID NOs:24-30 and 70.